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B3.0 > Advances in Animal Regeneration > Flashcards

Advances in Animal Regeneration Flashcards

1
Q

Structure

A

1.
2.
3.

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2
Q

Regeneration correlates with

A

stem cell potency

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3
Q

Planarians

A
  • lophotrochozoa
  • bilaterians
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4
Q

Planarian adult stem cells - the basics

A
  • Piwi+ (synapomorphy)
  • highly distributed
  • highly proliferative
  • multipotent (brain, eyes, gut, protonephreaia)
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5
Q

Piwi

A

germline marker

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6
Q

Schistosomiasis

A

stem cell proliferation drives liver stage

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7
Q

Planarian adult stem cells - the actual cells

A
  • mostly cytoplasm
  • perinuclear chromatic body (w/ RNA-rich granules)
  • protein components ~ germline
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8
Q

Planarian anatomy

A
  • 2VNCs
  • gut
  • 52 cell types
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9
Q

Planarian “immortal” life history

A
  • somatic
  • (a)sexual reproduction
  • one planarian soma has given rise to entire clonal strains
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10
Q

Planarian asexual reproduction

A
  • fissionates give rise to whole-body regenerated worms
  • cheaper to lose sex (liable to Muller’s ratchet)
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11
Q

Planarian sexual reproduction

A
  • hermaphroditic copulation and cross-fertilisation gives rise to polyembryonic cocoons
  • cocoons: many hatchlings
  • hatchlings develop sexual organs: juveniles
  • juveniles mature into adults
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12
Q

How do stem cells reach wound sites?

A

ongoing repair of migration-coupled DNA damage

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13
Q

IR

A
  • kills diving cells
  • depletes stem cells (recover)
  • apoptosis induced under breakage
  • planarians have moderately high R
  • 15 Gy
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14
Q

20 Gy

A
  • expression too high
  • stem cells persist but expression is non-functional post-3 days
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15
Q

Rad51

A
  • focal @ DSBs
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16
Q

PARP

A
  • focal @ SSBs
  • acute damage marker (~5 mins)
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17
Q

S. mediterranea transcriptomics

A
  • good complement of DNAR enzymes
  • redundancy and compensation
  • no BRCA1, XLF, XRCC4
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18
Q

Describe the redundancy and compensation of S. meditteranea transcriptomics

A
  • 1x pathway KO: functional
  • 2x pathway KO: stem cell depletion
  • IR + RNAi = toast! (biiiig decrease in stem cells; a lot tend to die/not recover)
  • quantify/count cells in brca2, rad51, parp1/2/3
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19
Q

Functional DNAR is necessary for

A
  • IR recovery
  • migration (<0.05)
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20
Q

Which DNAR transcripts does S. mediterannea present?

A
  • RPA
  • RAD51
  • XPF
  • ERCC1
  • PARP1/2/3
  • XRCC1
  • Pol Q
  • Ku70
  • DNA-PK
  • Artemis
  • Lig IV
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21
Q

Stem cell migration in planarians

A
  • decapitation: highly directional migration specifically to wound
  • poke the midline: migration
  • notch on midline: stem cells precisely veer there
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22
Q

Genetic toolkit underpinning migration

A
  • candidate gene approach (look @ migration TFs in other systems: EMT)
  • RNAi conserved TFs
  • migration decreased
  • necessary
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23
Q

microcapillary cell migration

A
  • generates mechanical nuclear stress: deformation
  • DNA damage
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24
Q

Give some processes that give rise to genomic misorganisation under micro capillary cell migration

A

i) mechanical shearing
ii) DNA repair factor exclusion
iii) cytoplasmic nuclease entry
iv) chromatin fragmentation
v) chromatin herination
vi) organellar mislocalisation

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25
What are the ultimate causes of genomic misorganisation under micro-capillary cell migration?
- epistatic and gene stability effects - lead to cancer and ageing
26
stem cells
- highly migratory - survive constriction (p <0.05)
27
how can constriction be quantified
- nuclear aspect ratio
28
Migration-coupled repair
- ^ SSB repair - ^DSB repair - Commet assay; independent of IR (migration-specific)
29
What is the relationship between migration and damage?
- pre-migration IR load: 5/10Gy at different time points - migration is paused/delayed - less distance covered (<0.05Gy, <0.01 10Gy)
30
Does migration sensitise to IR?
i) apply dose during migration ii) plot distance against sensitivity: correlation - damage is a significant load on machinery
31
Adult stem cells in acoelomorphs
- lots! - Piwi+
32
Aceolomorphs
- regenerative model - similar patterns to Planarians - not as powerful - e..g Hofsteria miamia (3-banded panther worm)
33
Adult stem cells in Cnidaria
- clonal - Piwi+ - migratory - e.g. Hydractinia
34
Why is it interesting to study zebrafish hearts?
- stem cells are involved in repair and immunity - physical function of heart disease - biomedical engineering
35
Describe the zebrafish heart
- main ventricle - cardiomyocytes - vesicles: blood supply - epicardium - fibrin
36
epicardium
strong, stable
37
What happens on injury to human hearts?
epicardial tears form scar tissue which blocks blood flow
38
Danio rerio heart regeneration
- injury is resolved w new cardiomyocyte remodelling - function restored after 30 days - no damage after 60 days
39
experimenting on heart
- brute force cuts (not replicative of reality) - cold probe - genetic ablation (mimics heart disease) - temporary hypoxia reoxygenation (mimics coronary vessel blockage; realistic, refined) - lineage tracing (cellular contributions to regeneration) - transcriptomics
40
what happens when you make brute force cuts on human hearts?
- loss of blood flow - hypoxia - crisis (part of the heart muscles loses function) - necrosis - failure
41
cold probing D. rerio hearts
- necrosis cleared - replacement cardiomyocytes observed
42
genetic ablation of D. rerio hearts
- create a genetic mosaic of ablation susceptibility - ablate - functional + necrotic mix
43
lineage tracing
i) recombinase + tissue specific promotor + drug (nt mimic; activates recombinase) ii) activates fluorescent protein in sp. cells (e.g. muscle + GFP cassette)
44
How to investigate epicardial gene expression
i) brute force heart resection ii) epicardial cell sorting (GFP+) iii) RNA-Seq iv) ATAC-Seq
45
ATAC-Seq
- chromatin state - ORF sequence - TF binding motifs - helix shape for binding, recruitment and interactions - generate dynamic time course tornado plot
46
wt1
- key epicardial injury TF - candidate gene - correlate RNA + ATAC-Seq
47
Interpreting ATAC-Seq
- enriched gene ontology: linkage - look for enriched TF motifs - build hypothetical reciprocal interactions to elucidate the regeneration-activated network - verify by in situ hybridisation
48
testing enhancer activity
- make transgenic lines - are they expressed in the right epicardial tissue?
49
life-history dependent regeneration
- frog legs - mouse fingers
50
context-dependent regeneration
- animals that don't regenerate well, except @ an early developmental stage - powerful experimental systems
51
tadpoles
- complex tail - amputation: complete regeneration; stage-determined - N40-46: yes - N46-48: no - N48+: yes - N52-58: slow decline - post-metamorphosis, adult limbs cannot regenerate
52
mice
- distal tip regeneration into adulthood
53
distal tip regeneration
most animals cannot do this post-neonatally
54
xenopus resection biases
- joint vs bone bias - distal vs proximal bias (to the main body wall)
55
mouse resection bias
distal-proximal bias
56
Adult Xenopus laevis wearable bioreactor set-up
- biodome + silk mesh (+plastic) - delivers acute key multi drug chemicals that innervate different signals over 24hrs - monitor for 1.5 years - control: just biodome - facilitates long-term regeneration and functional recovery
57
Adult Xenopus laevis wearable bioreactor obs - spike and soft tissue
- nothing: spike - biodome: more spike - biodome + drug: MORE spikes + soft tissue length (p<0.01)
58
Biodome effects on bone regrowth
- under CT - bone volume sig ^ (p<0.05)
59
Adult Xenopus laevis wearable bioreactor obs - wounding delay
- nothing: fibrin scar, barrier - biodome: increased diameter - treatment: heavily increased diameter (p<0.005)
60
Adult Xenopus laevis wearable bioreactor obs - gene expression
Yamanaka factors: Sox2
61
Animal models to study regeneration
1. Mus 2. Xenopus 3. Danio 4. Planarians 5. Acoelomorphs 6. Anolis 7. Hydra 8. Axolotls
62
Xenopus
- transgenesis - CRISPR-Cas9 - morpholinos - grafting/transplantation; cell fate - live imaging (early tadpoles = transparent) - RNA-seq (bulk and single cell)
63
morpholinos
- -ve sense RNA; KO in early dev
64
Danio
- stable transgenesis - CRISPR-Cas9, TALENs - lineage tracing - single cell RNA-seq - live imaging (transparent larvae + adults) - optogenetics - ATAC-Seq, Chip-Seq
65
Mus
- CRISPR-Cas9 - transgenesis - lineage tracing - single cell RNA-seq - spatial transcriptomics - organoids - in vivo imaging
66
Planarians
- RNAi (feeding/injection) - in situ hybridisation - single cell RNA-Seq - limited transgenesis
67
Axolotl
- CRISPR-Cas9 - transgenesis - bulk + single cell RNA-Seq - grafting - fate mapping - live imaging (limited to early stages due to pigmentation)
68
Anolis
- histological + immunological analysis - RNA-seq - genome sequencing - emerging CRISPR
69
Hydra
- transgenesis - RNAi + morpholinos - live imaging (transparent) - single cell RNA-Se1
70
Notophthalamus viridescens context
- adults: regenerate lenses - transdifferentiation of PECs in the dorsal iris - dedifferentiation and cell cycle entry
71
PECs
pigment epithelial cells
72
cdks
- regulated by cdkIs - control G1 progression
73
CDKIs
- 2 classes: INK4s, Kip
74
Rb
- retinoblastoma protien - activate when hypophosphorylated: lens fibre differentiation - inactive when hyperphosphorylated: lens fibre apoptosis - p53-independent
75
advances in Notophthalamus viridescens regeneration
- primary PEC cultures inhibited in vitro - cdk2 selective inhibition in vivo - necessary for lens regeneration by decreasing cell proliferation and increasing apoptosis
76
Eya2
- eyes absent 2 - redundant regulatory component of the DNA damage response in Ambystroma mexicanum
77
advances in Ambystoma mexicanum regeneration: Eya2
- functional ablation by acute pharmacological inhibition: Eya2, DNA damage signalling is dysregulated, esp. during limb regeneration @ proliferating progenitors - G1, G2 transitions impaired - decreased regeneration rate - no accumulation of DNA damage (compensation) - verified by double mutant RNA-Seq
78
advances in Ambystoma mexicanum regeneration: Chk1/2
- inhibition: severe regeneration impairment
79
Advances in Neodermata regeneration
Neodermata are capable of regeneration under xenobiotic stress/immune attack
80
Neodermata
- adult schistomes contain neoblasts - unlikely to experience amputation stress
81
Neodermata expt: immunity
- PQZ treatment @ sub-curative doses: severe tegumental damage (surface, cell bodies, underlying mesenchymal tissues) - these can regenerate
82
Neodermata expt: fertility
- male worm absence results in small female worms w/ underdeveloped reproductive organs - hypotrophic reproductive organs regenerate on male presence
83
Nerodermata case study
- what allows these obligate parasites to thrive? - can we use this knowledge to tackle PQL R, for example by targeting neoblasts?

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