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ANAT 381 > Ryan Lecture 1 > Flashcards

Ryan Lecture 1 Flashcards

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1
Q

what do animal models let us do

A

understand biological phenomena (manipulate cells and groups and their connection to other tissues)
Distinguish between genetic and environmental contributions to a disorder/phenotype
Explore causes and treatments for human diseases (doing similar experimentation on humans either unethical or not feasible)

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2
Q

What to consider when choosing a model system

A

Evolutionary conservation of pathways, genetic info,etc (understand basic phenotype, more complicated models = more complicated it is)
Accessibility = is it amenable for manipulation (to study in dev,)
Timing of development of organ under study (accessible)
Short vs long life cycle (most = 2–>21 day gestation period, drosophila and worms = shorter generation time, chickens = lay fertilized eggs)
Techniques for genetic manipulation (available , enough material)
Size
Generation time (can afford to house and study animal, consider for grad student, grant money)

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3
Q

Do embryos resemble adults

A

No
Period of dev, does not resemble adults
Fetal development = looks more like adult

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4
Q

How does a single egg become a complex multicellular 3d organisms

A

Process requires =
Generating diff cell types (10^14 in mammals, diff types of cells, mainly in brain)
Assigning them diff shapes and functions
Patterning throughout time the embryo in 3 dimensions (know orientation early on, to organisms and where in tissues)

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5
Q

What are the 3 components to building embryos

A

Cells
DNA
Patterning

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6
Q

Describe cells - building embryos

A

Gametes
Zygotes (give rise to diff cells then=)
Tissues

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7
Q

Describe dna - building embryo

A

Genome - within
Epigenome - epigenetic chances
Variation - enhancers

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8
Q

Describe patterning - building embryo

A

Signalling
Mechanical forces - shaping early embryo
Environment= if restricts =can have defects -go awry
Expressed in diff number or shapes of cells - this matters

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9
Q

Define patterning

A

The developmental mechanisms (any and all) that cause cells that are initially equal to acquire different identities

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10
Q

Define induction

A

Process where signals from a cell or group alter fate of another (different) cell
- inducing signals

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11
Q

Define competence

A

Ability to respond to inducing signal (has potential to change fate)
Cells may be competent to respond to >1 signal
If not competent = no change = incompetent
Important for patterning of tissues

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12
Q

What is an important part of development

A

Cell death

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13
Q

Name the stages of cell potential

A

Totipotent
Pluripotent
Multipotent
Unipotent

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14
Q

Compare pluripotent and multipotent cells

A

Pluripotent = uncommitted cells
Multipotent = committed cells (to a limited subset cells)

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15
Q

Describe totipotent cell potential

A

Gives rise to anything
Any cells, eventually become unipotent
Single cell to 8 cell morula (mouse embryo), can use for genotyping

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16
Q

Describe pluripotent cell potential

A

Blastula stage - mouse embryo
Te cells = support embryo
Icm = embryo itself

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17
Q

Describe multipotent cells

A

Grown out laterally = limb bud ex
Gives rise to diff cells in arm

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18
Q

Describe unipotent cell potential

A

Specific function = cells WILL NOT change fate in response to signals

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19
Q

Describe cell commitment

A

Specification —> determination —> differentiation

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20
Q

Describe specified cell

A

Follows a specific pathway if left alone
But fate is flexible
Can respond to other signals - can become smothering diff if change position

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21
Q

Describe determined cell

A

Fate irreversible
Can no longer change its fate in response to new signals
Know what it will become, can still respond to signals but wont become something else

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22
Q

Describe a differentiated cell

A

Mature and specialized
Final developmental stage
End of a lineage
At end of pathway = final function

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23
Q

Describe cell A

A

Committed to a specific differentiation pathway in absence of signal
If differentiation of cell A can be altered in response to signal = cell is specific but not determined
It is competent to respond to the signal

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24
Q

Describe Cell B

A

Differentiation of cell B cannot be altered in response to a signal then cell B is said to be determined = not competent to respond to the signal

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25
Name the 3 types of specification
Autonomous Syncytial Conditional
26
What species use autonomous specification
Most invertebrates
27
Describe autonomous specification
Cells develop according to early fate Asymmetric distribution of cytoplasmic content (tfs, proteins, have diff info - between daughter cells) Usually in embryos with invariant cleavages If blastomere removed - cannot be replaced (remaining cells cannot compensate, will lack structures removed)
28
Describe ex of autonomous specification in tunicate - sea squirt
Each blastomere already contains positional info - if remove cells = others wont compensate Already specific fate - ectoderm, endoderm, mesenchyme, muscle, notochord Set from very beginning - not by gastrulation, like usual in embryo
29
Describe asymmetric cell division in 1 cell c elegant embryo
From first division 2nd division = more symmetric Labelled with gfp
30
What does autonomous specification depend on
Asymmetric cell division
31
Name the 2 things that happens during autonomous specification - division
1. Symmetrical parent cell becomes polarized due to asymmetric segregation of fate determinants (cell fate determinants segregates in diff cells) 2. Production of distinct daughter cells depends on alignment of mitotic spindle = need asymmetric localization and spindle to line up properly
32
Describe determination gradient
Asymmetric distribution of determinants = 2 poles = diff factors, but asymmetry depends on how cells divide - mitotic spindle Also could be random or cues within field of cells that would help dictate = polarity in field of cells
33
Describe how get identical daughter cells
Spindle perpendicular = 90degrees to determination gradient Metaphase plate parallel to determination gradient = symmetrical divison
34
Describe how get NON identical daughter cell
Spindle parallel to determination gradient Metaphase plate perpendicaulr to determination gradient = asymmetrical division
35
Describe asymmetric cell division of sensory organ precursor cell in drosophila embryo
Important for autonomous specification 2 cells already very different 2 daughter cells Pon - partner of numb - cell surface protein, segregates to one daughter cell Histone H2B
36
What species uses syncytial specification
Insects
37
Describe syncytial specification
Nuclear division without cytokinesis - cell division, many nuclei in common cytoplasm Exposed to gradients of cytoplasmic components - morphogens A-P patterning in flies After cellularization - conditional specification Many nuclei and proteins - expressed and localized = compartmentalize, so each get diff cells like gradient
38
Describe ex of syncytial specification
Overall size of embryo not changing Nuclear division without cytokinesis = syncytium Very rapid nuclear divisions in cytoplasm Diff factors in cytoplasm
39
What species uses conditional specification
Regulated Vertebrates
40
Describe conditional specification
Depends on environmental conditions = cell extrinsic signals External signals cause diff tfs to be turned on or become active, coming from outside cell, context dependent - regional - distance from signals Context dependent - neighbours matter Cells can compensate for missing cells (robust system, not like autonomous)
41
Describe single cell rna sequencing to monitor differentiation
Zebrafish Staged embryos —> dissociate —> single cell isolation —> scRNAseq —> gene expression space = compare mRNA patterns of each cells, analyze clusters of expression - diff transcription patterns in cells
42
Describe experiment to monitor differentiation - what is seen at each stage
Experiment starts at beginning of gastrulation = almost all cells epiblast Gastrulation gives = ectoderm (epidermal), mesoderm and endoderm Next see differentiation of ectoderm info neural ectoderm (first get germ layer that starts to differentiate more)
43
Describe experiment to monitor differentiation -gen
4 hours post fert= see early stage gastrulation, mesoderm and ectoderm, and then later differentiation of neuroectoderm See many types of cells Dynamic process - rapidly turning on patterns of gene expression and then turn off fast - short period
44
Describe find it lose it move it
Expression of gene - bulge in embryos Cut it out and remove it treat with something to kill cells or genetic knockout Hoe does cell react to different environment, do they still become same cells - are they determined, what is being expressed, what cues
45
Describe find it lose it move it ex
Chick embryo - asymmetric expression on one side of embryo Important for determining right and left sides Move and see what happens
46
Describe specific ex of find it lose it move it
When red cells are removed from the blastula and placed in culture they become muscle cells When yellow cells are removed from blastula and placed in culture they become neurons In these 2 specific locations
47
Describe specific ex of find it lose it move it What happens when you take a red cell and mix it with many yellow cells????
= red cell becomes muscle, yellow cells are neuron = red cell becomes neuron, yellow cells are neurons - how we do explain - Mix and match = consider competence, inducing, quantities
48
Describe specific ex of find it lose it move it - what can we interpret from experiment
Take cells from blastomere and Transplant to diff embryo location = fate of neighbours or was it already determined, will it die
49
Describe early steps in vertebrate lens formation
Lens placode and neural ectoderm = neural ectoderm induces lens placode 2 way convo - feedback , change shape of cells - morphogens, lens pushes in and neural ectoderm indents Induction to form lens
50
What is lens placode
Thickened region of cells - ectoderm cells, of lens
51
Describe in situ hybridization to identify cells expressing target mRNA
RNA made complementary to specific mrna= labelled antisense with tag on utp Synthesize antisense probe and soak embryo and will bind to sense strand Wash out anything unbound and throw in antibody Alkaline phosphatase - conjugated antibody to digoxigenin = binds to dig, that labels utps Becomes coloured when phosphate removed
52
What is expressed in optic vesicle
Fgf8 Defined area, in specific region of neural tissue
53
Describe experiment to look at role of the optic vesicle in lens induction
What happens if move fgf8, = does it induce other tissues to become lens (is tissue competent to respond to fgf8) Make fgf8 bead -a nod see if induces pattern - monitor expresssion of gene expressed in lens
54
Describe experiment to look at role of the optic vesicle in lens induction - results
If cut out = do not from lens Is move = do not induce lens, so region not competent to respond to fgf signals - no signal = no thickening = no placode
55
What are inductive signals
Something Need 2 things = to produce and somethings to receive= signalling cell and receiving cell l
56
Describe signalling cell
Ligands
57
Describe receiving cell
Receptors = bind Ligand, received protein or cell Transduction mechanism = receptor to nucleus or actin cytoskeleton = need way to take signal and transducer into interpretable message Response = change in gene expression, cell shape, movement,
58
Name all steps of signalling
1. Signalling cell 2. Receiving cell needs receptor 3. Transduction = usually changes in gene expression 4. Response = can be subtle or could move away, further step in differentiation
59
Name and describe the 4 types of signalling
Autocrine = produced by itself, same cell responds to its own signal Endocrine = usually produced by tissues distances away, ligand travels via bloodstream Juxtacrine = directly between 2 cells Paracrine = most used, secreted from cell and reaches target cell
60
Compare juxtacrine and paracrine
Paracrine = some distance If response depends on = number of receptors, how much ligand secreted, or hoe much cells secrete, ligands ability to move
61
Describe ex of endocrine signalling
Retinoic acid pathway Small molecule travels through blood stream Retinol receptor = stra6 = ALSO a TF When bind = translocated to nucleus and bind dna and changes expression of genes
62
Describe ex juxtacrine signalling
Notch delta signalling - cell surface proteins Ligand delta/jagged for notch = need direct contact Notch held on membrane but also extra cellular space Get cleavage and then notch intracellualr domain goes into nucleus Need direct contact between proteins of the 2 cells
63
Describe cell-cell juxtacrine signalling through gap junctions
Direct communication exchange info Ions & small molecules go through Can set up gradients
64
What Is notch delta signalling used for
Compartments and boundaries
65
What is Juxtacrine signaling through jap junctions used for
Left right patterning in chick embryo
66
Describe ex paracrine - bmp proteins
Huge fam tbfbeta 1. Ligand forms complex with type 1 and type 2 receptors 2. Type 2 R phopshorylated type 1 R 3. Type 1 R phosphorylated R-smad 4. PhosphoR-smad dimerizes with co-smad 5. Translocation of smad diner to nucleus 6. Activation of gene expression Heteromeric receptor and phophorylates = several diff smads, not all smads respond to same ligand Phosphorylation events ultimately phosphorylation a tf = closed to open and binds another tf —> translcoated to nucleus
67
Give ex of bmp protein signallings
Activin - Xenopus organizer Nodal - left right patterning Dpp- drop Sophia segments
68
Describe paracrine ex - sonic hedgehog signalling pathway
1. Shh binds to patched receptor - release inhibition 2. Relieves patches repression of smoothened - active tf 3. Smoothened activated gli transcription factor
69
Describe shh pathway inhibitor
Inhibitor of pathway= cylopamine = cause cylopic phenotype = close tissues that form between eyes
70
Give exs of shh pathway
A-P patterning in limb dev Left-right patterning
71
Describe paracrine signalling = fgf
All gave diff heteromeric receptor complexes Signalling cascade, many phosphorylation events Complex transduction = tfs enter = nucleus turns on gene expression If anything blocks = will not work
72
Name exs of fgf signalling
Limb development Somite formations
73
Describe paracrine signalling - wnt signalling pathway
Stabilization through cascade of beta catenin Enters nucleus and triggers gene expression
74
Give exs of wnt signalling
DV axis in xenopus A-P patterning drosophila - wg wingless
75
Describe fgf gradient
Diff effects on signal Fgf8 secreted Free diffusion of fgf8 = greatest distance travelled, fastest way = affects cells pretty far away Diffusion confined by hspg clustering = surface where fgf binds Hspg directed diffusion of fgf8 = moves in ecm across many cells, can also hold it up Hspg = each bind to own fgf and has own response Fast or slow degradation of mrna affects protein level
76
Name the different kinds of ligands
Ligands that diffuse directly into cell Ligands that bind to cell surface - TM - receptors morphogens
77
Give ex of morphogens that diffuse directly into cell
Retinoic acid Glucocorticoids Estrogen
78
Describe ligands that diffuse directly into cell
Ligand bound receptor acts as tf to modify patterns of gene expression
79
Describe ex of ligands that bind to cell surface receptors
Fgfs, bmps, shh, wnt
80
Describe ligands that bind to cell surface
Must consider = source and how they get to target = what environment like, dense or loose cells Intracellular portion of receptor triggers downstream signalling cascade that activates changes in gene expression or cell behaviour
81
Describe morphogens
Can trigger v diff response in same cell = based on dose = concentration Secreted ligands that cause different/distinct responses at different distances/concentrations
82
Give ex of morphogens
All morphogens are ligands except biocid Shh, some bmps, ra, bicoid (tf, not secreted)
83
Name types of morphogens and give ex of each
Signalling molecules = shh, bmp/dpp (tgf beta super fam) Transcription factors = bicoid Small diffuse molecules = Retinoic acid
84
Describe characteristics of morphogens
Ligands that is secreted from localized source Forms concentration gradient Causes different/distinct responses at diff distances/concentrations Each cell sees a diff concentrator Activates target genes above a concentration threshold - depends on threshold
85
What to consider = how morphogens move
Size of molecule Type of tissue Fast vs slow dev
86
Name modes of movement of morphogens
Simple diffusion Restricted diffusion (concentration of cell surface receptors) Hspgs Transcytosis (between cells) Lipoprotein particles
87
Describe hspgs
Heparin sulphate proteoglycans Glypicans binds to and stabilize morphogens Helps morphogens move by dissociating and reassociating with hspgs

ANAT 381 (24 decks)

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