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ANAT 381 > Ryan Lecture 1 > Flashcards

Ryan Lecture 1 Flashcards

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
Q

Name the 3 types of specification

A

Autonomous
Syncytial
Conditional

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26
Q

What species use autonomous specification

A

Most invertebrates

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27
Q

Describe autonomous specification

A

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)

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28
Q

Describe ex of autonomous specification in tunicate - sea squirt

A

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

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29
Q

Describe asymmetric cell division in 1 cell c elegant embryo

A

From first division
2nd division = more symmetric
Labelled with gfp

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30
Q

What does autonomous specification depend on

A

Asymmetric cell division

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31
Q

Name the 2 things that happens during autonomous specification - division

A
  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
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32
Q

Describe determination gradient

A

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

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33
Q

Describe how get identical daughter cells

A

Spindle perpendicular = 90degrees to determination gradient
Metaphase plate parallel to determination gradient
= symmetrical divison

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34
Q

Describe how get NON identical daughter cell

A

Spindle parallel to determination gradient
Metaphase plate perpendicaulr to determination gradient
= asymmetrical division

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35
Q

Describe asymmetric cell division of sensory organ precursor cell in drosophila embryo

A

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
Q

What species uses syncytial specification

A

Insects

37
Q

Describe syncytial specification

A

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
Q

Describe ex of syncytial specification

A

Overall size of embryo not changing
Nuclear division without cytokinesis = syncytium
Very rapid nuclear divisions in cytoplasm
Diff factors in cytoplasm

39
Q

What species uses conditional specification

A

Regulated
Vertebrates

40
Q

Describe conditional specification

A

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
Q

Describe single cell rna sequencing to monitor differentiation

A

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
Q

Describe experiment to monitor differentiation - what is seen at each stage

A

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
Q

Describe experiment to monitor differentiation -gen

A

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
Q

Describe find it lose it move it

A

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
Q

Describe find it lose it move it ex

A

Chick embryo - asymmetric expression on one side of embryo
Important for determining right and left sides
Move and see what happens

46
Q

Describe specific ex of find it lose it move it

A

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
Q

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????

A

= 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
Q

Describe specific ex of find it lose it move it - what can we interpret from experiment

A

Take cells from blastomere and Transplant to diff embryo location = fate of neighbours or was it already determined, will it die

49
Q

Describe early steps in vertebrate lens formation

A

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
Q

What is lens placode

A

Thickened region of cells - ectoderm cells, of lens

51
Q

Describe in situ hybridization to identify cells expressing target mRNA

A

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
Q

What is expressed in optic vesicle

A

Fgf8
Defined area, in specific region of neural
tissue

53
Q

Describe experiment to look at role of the optic vesicle in lens induction

A

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
Q

Describe experiment to look at role of the optic vesicle in lens induction - results

A

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
Q

What are inductive signals

A

Something
Need 2 things = to produce and somethings to receive= signalling cell and receiving cell l

56
Q

Describe signalling cell

A

Ligands

57
Q

Describe receiving cell

A

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
Q

Name all steps of signalling

A
  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
Q

Name and describe the 4 types of signalling

A

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
Q

Compare juxtacrine and paracrine

A

Paracrine = some distance
If response depends on = number of receptors, how much ligand secreted, or hoe much cells secrete, ligands ability to move

61
Q

Describe ex of endocrine signalling

A

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
Q

Describe ex juxtacrine signalling

A

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
Q

Describe cell-cell juxtacrine signalling through gap junctions

A

Direct communication exchange info
Ions & small molecules go through
Can set up gradients

64
Q

What Is notch delta signalling used for

A

Compartments and boundaries

65
Q

What is Juxtacrine signaling through jap junctions used for

A

Left right patterning in chick embryo

66
Q

Describe ex paracrine - bmp proteins

A

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
Q

Give ex of bmp protein signallings

A

Activin - Xenopus organizer
Nodal - left right patterning
Dpp- drop Sophia segments

68
Q

Describe paracrine ex - sonic hedgehog signalling pathway

A
  1. Shh binds to patched receptor - release inhibition
  2. Relieves patches repression of smoothened - active tf
  3. Smoothened activated gli transcription factor
69
Q

Describe shh pathway inhibitor

A

Inhibitor of pathway= cylopamine = cause cylopic phenotype = close tissues that form between eyes

70
Q

Give exs of shh pathway

A

A-P patterning in limb dev
Left-right patterning

71
Q

Describe paracrine signalling = fgf

A

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
Q

Name exs of fgf signalling

A

Limb development
Somite formations

73
Q

Describe paracrine signalling - wnt signalling pathway

A

Stabilization through cascade of beta catenin
Enters nucleus and triggers gene expression

74
Q

Give exs of wnt signalling

A

DV axis in xenopus
A-P patterning drosophila - wg wingless

75
Q

Describe fgf gradient

A

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
Q

Name the different kinds of ligands

A

Ligands that diffuse directly into cell
Ligands that bind to cell surface - TM - receptors morphogens

77
Q

Give ex of morphogens that diffuse directly into cell

A

Retinoic acid
Glucocorticoids
Estrogen

78
Q

Describe ligands that diffuse directly into cell

A

Ligand bound receptor acts as tf to modify patterns of gene expression

79
Q

Describe ex of ligands that bind to cell surface receptors

A

Fgfs, bmps, shh, wnt

80
Q

Describe ligands that bind to cell surface

A

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
Q

Describe morphogens

A

Can trigger v diff response in same cell = based on dose = concentration
Secreted ligands that cause different/distinct responses at different distances/concentrations

82
Q

Give ex of morphogens

A

All morphogens are ligands except biocid
Shh, some bmps, ra, bicoid (tf, not secreted)

83
Q

Name types of morphogens and give ex of each

A

Signalling molecules = shh, bmp/dpp (tgf beta super fam)
Transcription factors = bicoid
Small diffuse molecules = Retinoic acid

84
Q

Describe characteristics of morphogens

A

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
Q

What to consider = how morphogens move

A

Size of molecule
Type of tissue
Fast vs slow dev

86
Q

Name modes of movement of morphogens

A

Simple diffusion
Restricted diffusion (concentration of cell surface receptors)
Hspgs
Transcytosis (between cells)
Lipoprotein particles

87
Q

Describe hspgs

A

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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