1
Q
What are three key cellular processes that contribute to morphogenesis?
A
Cell Adhesiveness, Cell Shape, and Cell Motility
- Cell surface proteins determine specificity and strength of adhesion
- Cytoskeletal proteins are responsible for cell motility and shape
- Patterning genes (homeotic genes, transcription factors) control spatial expressions of molecules (gradients) involved in adhesion and motility
2
Q
What do cell sorting experiments tell you about the adhesion property of cells?
A
- Two pieces of early ectoderm from an amphibian fuse to form a ball of tissue but endoderm and ectoderm will separate
- When presumptive mesoderm is mixed with presumptive ectoderm, the cells will group together
3
Q
Name adhesion molecules involved in morphogenesis.
A
Adheren Junctions:
- Actin bundles have cateninsbound and cadherins
- Depend on Ca2+
Desmosomes:
- Intermediate filaments have desmoplakin and plakoglobin bound which holds **cadherins*
- Dependent on Ca2+
Calcium independent adhesion:
- Involved the immunoglobin superfamily N-CAM
Cell Matrix Adhesion: (ECM)
- *Actin bundles** have vinculin and talin bound which hold integrin in the extracellular matrix to bind glycoproteins (laminin)
4
Q
How do cadherins function in adherin junction? How about in desmosomes?
A
- Cadherins provide adhesive specificity
- Different types of adherins (E,P,N-) will segregate where each cluster will be a specific type of cadherin
5
Q
Where do you expect to see actin bundles and intermediate filaments?
A
Under the plasma membrane
6
Q
How do CAMs Function?
A
Cell Adhesion Molecules are a part of immunoglobin superfamily
- Bind via homophylic interaction (CAM to CAM)
- Ca2+ independent
7
Q
How does the amount of Cadherin protein expression affect cell adhesiveness?
A
- The same cadherins aggregate with the same type even if they start off mixed randomly
- If there is more cadherins, they will be closer to the center
8
Q
How do integrins work?
A
- They sit in the extracellular matrix and can bind glycoproteins for cell signaling reasons
- Independent of Ca2+
9
Q
What are extracellular matrix proteins?
A
- Proteins in the ECM that result in the adhesive protperties of cells
- Examples include Calherins, integrins, laminin, fibronectin, proteoglycan, and collagen
10
Q
List 4 types of extracellular matrix proteins. Why are these proteins important for morphogenesis.
A
Laminin: Major proteins in basal lamina
Fibronectin: High MW glycoproteins that bind to integrins
Proteoglycan: Heavily glycosylated protein
Collagen: Composed of triple helix. main structural protein of various connective tissue (25-35%)
11
Q
Name the Adhesion molecules that are expressed in preimplantation mammalian embryos.
A
- Tropoblasts of the embryo express E and P Cadherins that bind to integrin receptors allowing implantation resulting in the embryo binding to the uterus
12
Q
What are polarized and non-polarized cells? What do they become?
A
Polarized:
- Gives rise to trophectoderm
Nonpolarized:
- Give rise to non-polarized cell
13
Q
What does E-Cadherin do during preimplantation mouse embryo development?
A
- E-cadherins are initially expressed everywhere but become restricted to intercellular contact regions at 8 cell stage. This will promote compaction
- Activation of E-cadherin signaling before the 8 cell stage results in premature compaction
14
Q
What is apical constriction? Where do you see this during neural tube formation?
A
- Hinge points at the medial and dorsal lateral regions that have a lot of myosin resulting in shape change to lead to neural tube
- Neural tube formation starts 19 hours after the egg is laid and ends 26 hours after the egg is laid
15
Q
How do trunk neural crest cells migrate to form sympathetic ganglions?
A
- NCC migration is dependent on the expression of cell surface molecules
- Dorsolateral -> region under ectoderm becomes skin and feathers
- Ventral: NCC moves into somites (dorsal root ganglia) to form sympathetic ganglia (adrenal medula, epinephrine, dopamine)
- Trunk NCC selectively migrates to anterior parts of somites
16
Q
What was the purpose of performing a nuclear transplantation experiment?
A
- To see if a differentiated cell contained all the genetic information needed to create an identical organism
17
Q
What is an enucleated egg?
A
- Removing the nucleus from an egg
18
Q
What was the outcome of the nuclear transplantation experiment performed by Briggs and King?
A
- Briggs and King concluded that nuclear transfer might not be able to reverse embryonic differentiation
19
Q
What modifications did Gurdon introduce to perform successful nuclear transfer?
A
- UV radiation was used to fragment DNA as opposed to using a pipette to stab oocyte
- Used Xenopus
20
Q
What was the general conclusion of Gurdon’s experiments?
A
- A cells ability to de-differentiate could be tested by placing a nuclei from a differentiated cell into an egg
- Nuclei from adult skin, kidney, heart, and lung can develop into an adult but show a lower survival rate
- Genes required for development are not permanently altered (deleted)
21
Q
How was the first mammalian animal cloning done?
A
22
Q
How was it proven that Dolly was a cloned animal?
A
- The lambs white fave was different from the black face of the surrogate mother in addition to the identical DNA to the sheep it was cloned from
23
Q
What are iPSC cells? How are iPS cells made?
A
- Induced pluripotent stem cells
- Made from embryonic cells and adult fibroblast cells in addition to Yaminka factors
- Helps avoid graft rejection
24
Q
What advantage do iPS cells have over traditional ES cells?
A
- ES from IMC
- iPS from somatic cells
- You don’t need a female to develop an egg
- Stem cells can be generated by the patient resulting in no immune rejection when implanting skin grafts and such
25
What are the 4 Yamanaka factors?
- Oct 3/4
- cMyc
- Sox2
- K1f4
26
Understand how enhancers regulate gene expression. Know the difference between
enhancers and promoters.
27
What is Waddington’s epigenetic landscape model?
- Early embryonic cells have higher potency
- Ability to switch fates decreases as cells develop
- Cell lineage and interactions in space and time dictate cell fate outcome
28
Understand the importance of histone modifications and their functions. e.g., H3K4me1, K3K27Ac, H3K27me1
- Histone moditfication controls chromatin formation which means they control transcription of genes
- Acetylation causes chromatin to open up **transcription**
- Methylation causes chromatin to close up **no transcription** (exception is meth of K4)
- H3K4me1: transcription ON
- K3K27Ac: transcription OFF
- H3K27me1: transcription OFF
29
What is DNA methylation and which residue is modified?
- Methylation of DNA = Adding methyl groups
- Residue modified : converts DNA (cytosine) --> 5 methylcytosine
- Occurs in promotors and enhancers
30
What kind of epigenetic modifications occurs around active and inactive genes?
Active:
- Enhancers and promoters have no methylation
- RNA pol 2 binds to promoter/gene body
Promoter: H3k4me3
Enhancers: H3K4me1 and H3K27Ac
Inactive:
- Promoters and gene bodies are methylated
- RNA pol2 cannot bind
Repressors: H3K9me3 and H3K27me3
31
How do Trithorax and Polycomb complexes regulate the expression of Hox genes in
specific parasegments?
Trithorax:
- promotes acetylation: allows transcription for Ubx gene in one parasegment
- Activators: H3K4me1, H3K27Ac
Polycomb:
- Promotes histone methylation blocking transcription of Ubx in other parasegments
- Repressor: H3K27me3
32
What is TAD? What does it tell us?
- Topologically associated domains
- Tells us if chromatin is unfolded, active, red. If folded, inactive, dark red
33
How does the chromosome region surrounding HoxA complex gradually open up during early mouse development?
- Chromatin is condensed but becomes unfolded. HoxA genes on chromatin can be expressed A ->P
- Wnt promotes unfolding of chromatin so enhancer becomes exposed
34
What are the similarities and differences between mouse ES and Epi stem cells?
Similarities:
- Express tf Nanog, Oct4, Sox2, Klf4
- Can form teratomas
Differences:
- ES cells can contribute to chimeras
- Epi cells cannot contribute to chimeras
- ES cells can differentiate into any cell
- Epi cells can differentiate into a lot of cell types
35
How does therapeutic cloning differ from reproductive cloning?
Therapeutic cloning:
- When the embryo is formed, ES cells are derived to generate different types of tissue
Reproductive cloning:
- When the embryo is formed it is implanted into the surrogate mother and the clone is born
36
How can sickle cell anemia disease in mice be completely cured by gene therapy?
- Skin cells are gathered and viruses are used to reprogram cells into ES like-iPS cells
- Genetically identical iPS cells are generated with the corrected mutation and differentiate into blood stem cells
37
How does type I and type II diabetes differ? What are hypo and hyper glycemia?
Type I:
Beta cells in the pancreas are targeted and destroyed by the body (from birth)
Type II:
beta cells become desensitized to insulin resulting in high blood sugar (genetic and lifestyle)
Hyperglycaemia:
- High blood sugar
Hypoglycaemia:
- Low blood sugar
38
What is transdifferentiation? What are advantages of using this approach?
- Transdifferentiation is a process in which non-beta cells, typically from the pancreas, are reprogrammed to transform into insulin-producing beta cells which can potentially be a treatment for diabetes
- A person's own cells can be used and no other donor is required
- Other beta cell transplantations require immunosuppresants but this is a simpler process
39
What are organoids? What are some limitations?
- Organoids are minature and simplified versions of organs/tissue from stem cells
- Limited in complexity, genetic and phenotypic variability, immune interactions, and problems scaling to therapeutic applications
40
What are teratomas?
- ES cells injected into adult mice instead of IMS of blastocyst results in development of tetatocardnoma which is a tumor under the skin which has different tissue such as hair teeth and bone.
41
What are two different strategies used for germline differentiation in fish/frogs vs mammals?
- Fish/Frog matternally derived germline determinants (PGC) via germ plasm
- Mammals: Cell to cell signaling determines germ line (PGC)
42
What are the advantages of having sexual preproduction
- Allows for genome mixing
- Creation of new genetic combinations/variants
- Genetically different offspring
43
Describe an experiment that showed the importance of pole plasm during germ cell development.
1. UV light was used to destroy Pole plasm cells ---> germ cells did **not** develop
- Pole plasm neede for germ cell development
2. Can take posterior pole plasm of a donor, inject it into anterior region of an acceptor and pole cells will develop. injected into posterior of another acceptor will result in identical DNA to embryo B
- Pole plasm can make germ cells
44
Describe an experiment that showed oskar mRNA is essential for germ plasm assembly and function.
- Oskar mRNA localized to the posterior end and bicoid is localized anterior end
- If bicoid is replaced with Oskar, Oskar mRNA will now be anterior in addition to posterior ---> germ cells develop both A and P ends
- Oskar plays important role in pole plasm development. Mutant = sterile
45
Where are mammalian primordial germ cells (PGCs) formed? How do they migrate to the conad?
- No germ plasm. Cell cell interactions only
1. Cells that will become PGCs are set aside in early development to become sperm or eggs (epiblast stage E6.5)
2. Cells get BMP signal from neighboring extraembryonic ectoderm to specify into PGCs and migrate through indgut to gonad
3. Once in gonads, cells will go through mitotic proliferation, meiosis, and differentiate into eggs or sperm
46
Describe all stages of oogenesis and spermatogenesis. Understand when cells enter mitosis and meiosis.
47
What organelles are present in mature sperm?
48
What is protamine used for?
49
How does species specific fertilization occur?
50
What is the order of fertilization events?
51
How does the blocking of polyspermy occur during fertilization? Describe fast and slow block.
52
Define imprinting. How can a gene be imprinted?
53
What is the difference between parthenogenesis and gynogenesis?
54
What is the difference between androgenesis and gynogenesis?
55
What is the difference between parental and maternal imprinting?
56
Understand the maternal imprinting of lgf2 in mice. (note sex of parent that passes allele)
57
How does trisomy occur?
58
When does imprinting occur during embryogenesis? When are imprinted patterns erased?
59
What are Turner and Klinefelter syndrome?
60
How do Wolffian duct and Mullerian duct differentiate from mesonephros?
61
What does Sry do?
62
How is Sox9 expression regulated by Sry, and also by Wnt4.
63
How is sex determined in drosophila? Why is sex lethal (Sxl) protein only made in the female fly? How does Sxl protein function?
- Sex in flies is determined by ratio of X chromosomes to autosomes and sxl protein
- XX -> slx protein during synctial blastoderm stage -> female
64
How is dosage compensation accomplished in drosophila, C. elegans and mammals?
- Dosage compensation = mechanism to ensure male and females get same amount of X chromosome encoded proteins
- Drosophila: male X proteins are doubled via acetylation
- C elegans nematoda: in F both X are partially repressed
- Mammals: in f, 1 X becomes inactivated
65
Describe how Xist functions during early mammalian embryogenesis?
- Both X in female are initially active in females but one inactivates. Will be reactivated during germ cell development
- Inactive X chromosomes make Xis transcripts (RNA) and will be off. Active X does not make Wist and will remain active in life
66
What is Barr body?
- Inactive X chromosome found in female somatic cell
Developmental Biology
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