1
Q
Human advantages
A
self reporting mutants
many of us
many cell lines we can do experiments on - CRISPR/CAS9 system = can edit genomic info
2
Q
Human disadvantages
A
no direct experimental access
fetal material hard to obtain
3
Q
Primate advantages
A
similar to humans, post natal development
genome sequence known
4
Q
primate disadvantages
A
expensive
ethical consideration
5
Q
dogs/cats/ferrets advantages
A
mammals with post natal development (immature visual system when first born)
genome sequence known
6
Q
dogs/cats/ferrets disadvantages
A
slow reproduction
little access to embryos
7
Q
rats and mice advantages
A
mammals (homologous brain areas to humans)
many mutants
8
Q
rats and mice disadvantages
A
embryogenesis occurs inside mother
slow development
9
Q
birds advantages
A
embryos accessible = better manipulation
similar neural circuitry to mammals; just arranged differently
10
Q
birds disadvantage
A
little genetics
fetal experiments still difficult
11
Q
amphibia advantages
A
vertebrates
rapid development
12
Q
amphibia disadvantages
A
hard to make transgenic animals
genetics just getting started
13
Q
zebrafish advantages
A
transparent embryos - optogenetics
rapid development
14
Q
zebrafish disadvantages
A
not as easy to knock out genes
less embryos than invertebrates
15
Q
flies advantages
A
fast development
genome sequence known
16
Q
flies disadvantages
A
small neurons, but can still use optogenetics
not vertebrates
17
Q
worms/ c. elegans advantages
A
simple
invariant cell lineages
18
Q
worms/c. elegans disadvantages
A
not vertebrates
poor external morphology
19
Q
Gordon experiment
A
- UV light irradiated an embryo so that the nucleus is removed while everything else is intact
- nucleus is taken from an EPITHELIAL cell and injected the nucles into the irradiated egg
- embryo allowed to develop; a full grown frog was able to form
This experiment tells us that all cells have genetic info to make a full organism; it’s what genes are being transcribed that differs between each type of cell
20
Q
Two parts of a gene
A
- regulatory region - acts as a switch for a gene
2. coding region - info needed to code mRNA to make the protein
21
Q
Transcription factors; types of TFs; rule for gene activation
A
- sequence specific DNA-binding proteins
- activators and repressors
- all activators must be present and all repressors must be absent
22
Q
DPP gene - what are its functions; what is it
A
makes the epidermis; it is an inhibitor - represses neural genes (more effective at doing this than turning on neural genes)
23
Q
Mesoderm - what body parts does it give rise to
A
heart, muscle, fat, blood cells
24
Q
Neuroectoderm - what body parts does it give rise to; what are the transcription factors present?;
A
- nervous system
epidermis of ventral nature - AS-C - a transcription factor that turns on neural genes
Brk - a transcription factor, repressor of dorsal epidermal genes
Sog - a secreted protein, repressor of DPP
Rho - a tf?; creates ventral epiderms
25
Epidermis - what body parts does it give rise to
?
26
Signals and receptors - two types of reception
1. signal to ligand reception - cell A makes the ligand, it diffuses out of cell A, then attaches to a receptor on cell B, which causes a response: a change in gene expression (changes something in regulatory region of genes)
2. tethered signal - The ligand is made in cell A and is still tethered to cell A when it binds to the receptor in cell B
27
Morphogens; what are they; how it affects nearby cells; how to keep the gradient
1. a transcription factor that is secreted and is present in a graded fashion (cells further away get less of it cuz the gradient decreases with distance)
2. different levels/concs. of factor result in different effects/responses
high concentration (closest to morphogen) = cells form into cell state A
medium = b
low = c
3. the source must be near a sink - like a bathtub; the sink keeps the gradient present; if the source fills the environment with morphogen and theres not drain present, then you will reach a steady state which is unwanted
28
Dorsal - what is it and where are the highest levels
a transcription factor in fly embryos; the highest levels are in the mesoderm, then neuroectoderm, and pretty much nonexistent in the nne
29
Twist and Snail - what are they? what activates it? (explain the dorsal binding site for Sna) where are they located? Mutants?
1. transcription factos - Sna is a repressor of neural genes (including Vnd, Ind, Msh), and Twi is the activator of mesoderm genes
2. high levels of dorsal, which is in the mesoderm; Sna has a dorsal binding site - but it is weak/low affinity, so you must have high levels of dorsal to occupy the Sna regulatory region and turn the gene on
3. Sna mutant = neural genes would grow in the mesorderm because this repressor is absent; Twi mutant = looks like snail mutant - cuz this activator is absent, and the default state of cells is neural - Twist is not there to activate mesoderm genes so these cells turn neural
30
Dorsal epidermis - What tf is absent here? How does this affect the fate of the area? What does this signal result in?
1. Dorsal is absent
2. absence of dorsal = results in expression of a morphogen - DPP or BMP (bone morphogenic protein, which is a category)
3. this signal results in activation of epidermal genes and repression of neural genes
31
Fly patterning - how is it initiated
It is initiated by intrinsic cues (like Dorsal) provided by the mother in the egg
32
Frog patterning - how is it initiated
It is initiated by both intrinsic cues (like VegT) and extrinsic cues (point of sperm entry)
33
The two main regions of a frog embryo; who made these?? where is sperm attracted
The animal region (which has pigment) and the vegetal region (no pigment); it is made by the mom, and the boundary is where sperm is attracted
34
The actions that come into play after sperm entry
1. sperm enters the egg at the animal region and near the equator (extrinsic cue!)
2. Latent dorsalizing factors migrate to the dorsal pole, then they become active - active dorsalizing factors
3. VegT is a TF (an intrinsic cue) that activates mesoderm inducing cells that migrate into animal region
4. The active dorsalizing factors create a gradient of B catenin, a morphogen; the gradient is highest on the top (dorsal side) and lowest on the bottom (ventral side)
5. the area where the active dorsalizing factors are, eventually turns into the Spemann Organizer and then the notochord
6. Spemann organizer secretes neural inducing factors, causing the left side to become neural; also secretes chordin (similar to sog); a diffusable factor that moves in the same neural area, this is above BMP; chordin inhibits BMP
7. Ingastrulation - cell movement in the mesoderm - it folds inward and switches the anterior-posterior pole
35
VegT - what is it? what does it do? is it mobile?
1. a transcription factor present in the vegetal region; stays in cytoplasm and cannot diffuse
2. turns on genes that encode mesoderm inducing signals (tells cells "make mesoderm") - these signals can diffuse out of the vegetal region; VegT also turns OFF genes that RESPOND to mesoderm inducing factors; in the vegetal region, these genes are turned off. since these inducing factors are able to migrate, they migrate on to the animal region, where mesoderm is able to form.
36
B catenin; what is it; how is the gradient distributed; what does it do
1. it is a morphogen
2. the gradient is highest in the dorsal area (at the top)
3. it determines how the mesoderm is subdivided
37
organization of frog embryo
from left to right: ectoderm, mesoderm, endoderm
| within the mesoderm from bottom to top: blood cells, heart, muscle, notochord
38
Neural Induction - how Sog and Brk stop DPP
DPP is made in cell A, and is released; when it reaches cell B, it could bind to the receptor on cell B; but Sog is there outside the cell to stop it from binding - Sog can save multiple cells
Brk is only inside the cell, and can stop DPP from changing gene expression in cell B; Brk can only save one cell
39
Sog/Brk mutant
DPP will be allowed to autoactivate below the epidermis, so epidermal genes will be turned on in the neuroectoderm and skin will grow here; neural genes will be turned off
40
DPP mutant
the epidermis will turn neural!
41
BMP's - their characteristics in regards to turning neural genes off and turning epidermal genes on
BMPS are more effective at turning neural genes off than turning EPIDERMAL genes ON
you need more BMP to turn on epidermal genes
42
Mangold - spemann experiment - what was the experiment? what was the outcome? what was the conclusion?
1. One spemann organizer of one embryo was grafted into the bottom mesoderm area of another embryo, and the fate of the donor embryo cells was trackable
2. a two headed frog was created - a duplicated neural axis
3. the grafted piece still became a notochord, but the cells nearby the grafted piece misbehaved and turned neural; the notochord was derived from the donor, but the nervous system is being derived from the host
the notochord must be providing neural inducing factors that converted nearby cells neural when they were supposed to turn to epidermis
43
animal cap experiment - what were the two different conditions? what were the results? what was the secondary experiment? what were the conclusions?
1. animal cap cut off and left to grow by itself - result was a cap of epidermal cells; animal cap cut off and placed next to spemann organizer tissue - result was a cap of neural cells: then they separated the cap and spemann organizer with a filter, and neural cells still grew..... Conclusion: spemann releases neural inducing factors
2. condition 1: the animal cap was broken apart from each other, put in a centrifuge tube, then placed on a plate to grow - neural cells!
condition 2: same as 1, except add BMP - got epidermal cells
condition 3: same as 1, except add BMP and chordin - got neural cells
3. therefore, the breaking apart of cells keeps them from communicating and receiving BMP from each other, so theyre allowed to turn neural; BMP is necessary and sufficient to supress neural fate; chordin can reverse this effect
44
fate map
where does a cell in a certain region of an embryo go, and what does it become?
45
determination
active manipulation of embryo to test how committed cells are to a certain course; not always accurate
46
community effect
if you take out a large group of cells and transplant them, they retain their original identity
47
two types of genes
maternal - only active in mother when egg is being developed (ex. Dorsal)
zygotic - come from both parents; function in embryo (ex. Sna, Twi, AS-C, Rho, Sog, Brk, DPP)
48
Neural Competence - within the NE;
Not all neurons are the same in the neuroectoderm; some cells become neurons because they are competent to do so (cells that are incompetent become ventral epidermal cells); The AS-C gene is important in determing neural competence; if you don't have the AS-C gene, then you cant become a neuron (the epidermis lacks AS-C, so it can't become neural); AS-C turns on genes that maintin their AS-C "turn on"; if a gene has its AS-C turned off then itll turn into an epidermal cell
49
three zones of NE in invertebrates? What are the 3 neural identity genes? vertebrates? what are they? How are the zones made?
invertebrates: (from bottom to top) Vnd, Ind, Msh
vertebrates: (from bottom to top) Nkx, Gsh, Msx
The CNS is broken up into 3 domains; these three are transcription factors that are required for establishing different neuroblast identities; they are neural identity genes
There is a certain amount of pre-existing pattern that exists that eventually turn into discrete domains that communicate with each other
50
What's DPP's main job in the NNE?
There are high levels of DPP in the NNE, which is enough to repress all types of neural genes from growing here, including Msh and Ind
51
Explain the gradients of DPP and Dorsal and how they affect what forms in each region
DPP has a gradient downward and acts like a morphogen - shuts off all neural genes in NNE; DPP gradient high in row 3, shuts off Ind, allows Msh to be in row 3 (the gradient of DPP is not strong enough to turn of Msh); the gradient in row 2 is lower, and Ind is strong enough to withstand DPP - so Ind grows in row 2)
Dorsal has a gradient upward - Dorsal is required for activation of both Vnd (high levels) and Ind (medium levels of dorsal activate ind), not Msh; it acts as a morphogen cuz higher levels are required for Vnd vs. Ind
Vnd represses Ind and Msh
Ind represses Msh
Msh is present everywhere. Just only expressed in row 3
52
Patterning/creation of rows; and what branch theyre called
Activation branch - present in NNE because DPP activated epidermal genes, which eventually make a pattern (creates 2 rows)
Repression branch - all of the NE; Sog and Brk repress DPP, and creates a pattern within their space (3 rows)
53
DPP mutant
Ind would grow in row 3; Msh and AS-C incorrectly expressed in dorsal epidermis
54
Ind Mutant
Msh would be present in row 2 and 3
55
Vnd mutant
Ind would be present in row 1; Msh would turn on too but Ind blocks it
56
Why is AS-C only present in the NE?
Why isn't there ind, vnd, and msh in mesoderm?
DPP from above blocks AS-C, and Sna from below blocks it
Sna also blocks vnd, ind, and msh
57
In a Vnd and Ind mutant
Msh would be present in all three rows - expressed incorrectly in rows 1 and 2
58
Why do invertebrates have ventral nervous systems and vertebrates have dorsal nervous systems?
Formation of a neural plate; the neural plate then folds inward and moves all the way to the ventral side, and this is where the nervous system forms
59
DPP/ lacZ experiment - what did they do? Results? Conclusion?
Fused a regulatory region of DPP with a coding region of lac z gene. Result was that the b gal protein was made which makes cells turn blue, and the epidermis was all blue
Conclusion is that the regulatory region is independent of the coding region. Even though the lac z regulatory region was absent, lac z was still able to form the protein even with a diff reg. region
60
Dorsal mutant
DPP will be turned on throughout entire cell. Suppresses neural genes so cell will be entirely epidermis
61
DPP and dorsal mutant/ DPP, SNA, vnd, ind mutant
Msh present in entire embryo. No vnd and ind cuz there's no dorsal
Msh present in entire embryo , also twist. Cell gets mixed signals and dies
62
Within the NE, how do cells choose to become ventral epidermal cells or neurons - what is the process called? How does it work? Name all of the factors involved
This is called neural inhibition, which works through the Delta-Notch signaling system.
Developmental Neurobiology
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