DB5

cell renewal is more ___ than tissue renewal + examples

• cell renewal is more common

• cell renewal examples

  • hematopoietic SC, hair/skin SCs, muscle SCs, germline niche SCs (sperm)

• tissue renewal example

  • liver (undifferentiated cell type with multipotency)

adults have limited regeneration potential compared to neonates i.e digit tips

D1-D7/neonatal mammal: can regenerate digit tips (distal not proximal)

unlock regeneration potential in mammals by looking at regeneration models that do ___ regen i.e

• zebrafish, salamander/newt, axolotl

• LIMB REGENERATION

salamander limb regeneration + whats in limb

• tadpole grows limbs via AER/ZPA/PZ

• embryo → development → limbs

• injury → LIMB REGEN in 90 days

  • ampu → wound epithelium → blastema/mesenchymal → functioning limb that’s scar free (knows when to stop growth)

• inside limb:

  • skin, bones, nerves, cartilage, connective tissue aka many diff cell types

LIMB REGENERATION process IN DEPTH (7)

0. cut limb/injury/amput

   1. bleeding, exposed tissue

1. formation of wound epithelium

   1. epithelial cells migrate to cover injury/stop blood loss

2. innervation:

   1. nerves secrete fgf + bmp important signals

   2. (as fgf/bmp required to initiate bstma formxn)

3. formation of Apical Epithelial Cap (AEC)

   1. thickened epithelium that secretes fgf (similar to AER in limb dev) for growth

   2. (as fgf/bmp required to initiate bstma formxn)

4. Dedifferentiation + Migration:

   1. mature cells dediff. and migrate to wound site

5. Blastema Structure Formation:

   1. requires: dediff. mesenchyme from mature tissue, acts like SC

   2. requires ECM remodeling

      1. remodel old tissue + activate cellular programing

      2. promote cell migr for blastema formation

      3. support prolif + dediff

   3. migr+prolif (fgf+bmp)

6. Patterning the Regenerated Limb

   1. early stage: ECM molecules regulate fgf signaling, control A-P id

   2. later stage: SHH, FGF gradients establish A-P polarity

Limb Regen Summary (5)

1. Injury/Amputation

2. Wound Epithelium Forms (later AEC)

3. Dedifferentiation + Migration

4. Blastema Formation

5. Patterning + Growth

what’s required for Limb Regen (4)

1. initiation of regen: AEC + neurons → fgf+bmp REQUIRED for initiating blastema formation

2. blastema REQUIRES

   1. ECM remodeling, dedifferentiation, migration+proliferation via fgf+bmp

3. ECM will

   1. remove old tissue

   2. help activate cellular reprogramming / dediff.

   3. support cell migr/prolif

4. limb patterning requires

   1. ECM molecules regulating fgf early, control A-P identity

   2. later: shh, fgf gradients to establish A-P polarity

during limb regen: 2 domains

1. wound epithelium = specialized alyer of cells forming cap over wound/freshly amputated limb, important for directing regeneration process

2. blastema = mass of undiff. cells derived from dedifferentiated tissues, form at cite of injury + regeneration capacity (2 key: migration, proliferation)

   1. mesenchyme cells undergoing dedifferentiation

   2. the blastema acts like SC, but has 1:1 directionality i.e skin dediff. into blastema mesenchyme must diff. into skin

   3. consider blastema multipotent, but individual cells are unipotent

Signaling Pathways in Limb Regen (what is involved) (4)

• remaining nerves secrete fgf + bmp

• AEC secretes fgf to instruct interior epithelial tissues

• ECM remodeling: MMPs (matrix metaloproteinases) increase → make ECM plastic

• Shh + fgf gradient in blastema

over time, blastema formation (within it: migration+proliferation)

• fgf + bmp responsible for migr/prolif

• A/P Prox/Dist Axes still exist in blastema

Summary: fgf, bmp, MMPs, shh

Signaling Gradients in Limb Regeneration vs Limb Dev

• wounded limb maintains A/P, Prox/Distal axes

• LR:

  • SHH high at posterior end / gradient

  • fgf high at anterior end / gradient

• LD:

  • ZPA secretes SHH to form pinky at high SHH/thumb low SHH

  • AER secretes fgf so PZ grows/prolif

Cooperative Signaling in Limb Regen + EXP

• bmp and fgf cooperate with each other to help facilitate LR

  • need both SP/necessary, one is NOT sufficient

  • bmp MT → no LR, fgf MT → no limb

• EXP: injure (required) skin on arm → graft beads containing fgf alone, bmp alone, fgf+bmp

  • fgf+bmp → created mini arm on arm

  • fgf+bmp sufficient in injury context to induce limb formation

Limb D vs R: KEY (4)

cell source: undiff. emb. progenitor cells vs dedifferentiated cells from mature tissue

key structure: limb bud (AER, ZPA signalling centers) vs. blastema

cell potency: multipot. emb. cells vs. unipotent lineage-resetricted progenitors with memory

patterning signals: fgf, shh, wnt, RA vs reuse many same pathways in injury dependent context

Limb D vs R: Other Features (6)

positional info: establish de novo during emb.genesis vs. re-establish from remaining tissue context

growth control: programmed dev. timing i.e bmp/stop branching (bmp -| fgf) vs. growth stops when correct size/shape restored

immune involvement: minimal involvement/dev. env. vs. strong imm/inj response required (Mϕ, inflammation)

• i.e salamanders have imm response that turns on then off so regen programs continue, humans just get inflamm. response → fibrosis/scarring

scarring: no scarring vs. regen. without scarring (in regen. species)

outcome: 1st formation of limb vs. replacement of lost limb

species context: all vertebrates w/ arms, legs vs. robust only in some species (salamander not mammals)

EXP: staining during limb regen to visualize limb, blastema

EXP: method to identify progeny of blastema cells? (what do they become) (5)

1. see nuclei accumulating/density at blastema

2. use lineage tracing approaches

   1. Crispr/Cas9 lineage labelling

   2. Cre-LoxP + singlecellRNAseq

   3. Crispr/Cas9 knock in

   4. transgenic lin tracing model i.e hulk axo

   5. integrated w. scRNAseq

Axolotle Model Organism + limb regen EXP

• genetically trackable + completely sequenced genome

• hard to grow at scale

• hulk axo that expressed GFP in entire body

EXP: lineage tracing

• transplant GFP cells from donor hulk axo → non-GFP expressed axo

  • graft GFP skin onto non-fluorescing limb

  • cut/amput limb

  • track fate + positional identity (what, where)

tissue-to-tissue mapping of LRegen (results of linage tracing)

key: lineage restricted

before amp —regen→ regenerated limb

dermis → dermis, dermis → skeleton

skeleton → skeleton, skeleton → dermis

• dermis + skeleton both from lateral plate mesoderm

muscle → muscle (presomitic meso)

schwann cells → schwann cells (neural fold)

epidermis → epidermis (lateral ecto)

fate mapping of blastula:

• ecto → skin (epiderm), CNS

• meso → somites (dermis, skeleton, muscles, brown fat), lateral plate mesoderm (limb, blood, heart), intermediate mesoderm (kidney, gonads)

• endo → organs / pancreas, liver

blastema conserved in other species i.e zebrafish + tail amputation/injury

• use single cell approaches

  • collect samples at diff time points, sequence, figure out what cell types inside

• amputate tail → 3 day regeneration

  • tail includes notochord

• not as good as axolotl

AER vs AEC (LD s LR) (3)

• apical ectodermal ridge secretes fgf // apical epithelial cap secretes fgf + bmp

• ZPA → shh // shh signaling but not via ZPA

• similar: both have fgf source, but different: one is AER vs AEC + nerves, same fgf molecule tho

regeneration in non-regenerating models

• from last universal common ancestor, deuterosomes, chordata, humans/Ms vs amphibians/fish

  • Ms low cap for regen, fish/amp high

• reactivate in Ms, etc., why do some regen. some dont?

  • ask the questions: why they CAN regenerate? what are barriers to regeneration?

Modes/Classes of Regen (3+1)

1. rearrangement of pre-existing tissues

   1. injury → rearrange remaining stuff

2. use of adult somatic SCs (hair, skin, blood)

3. dedifferentiation and/or transdifferentiation of cells

all involve

determination → differentiation → restablishment of proper scale, proportion

Classification/Animals (5)

a) whole body regen / planaria flatworm

b) structural regen / newt limb, zfish fin

c) organ regen / zfish parts of heart

d) tissue regen / blood SCs, drosophila gut lining

e) cellular regen / C elegenas axons

Cellular Regeneration i.e asym cell div

• asym cell div: HSC → 1 HSC, 1 differentiating cell (into Tcell, RBC)

• dividing cell into A and B

  • transdifferentiation: A→B, directly convert into another cell type

• transition state cell type: A→ C→ B

Planaria/flatworm defy aging + Neoblasts

• whole body regeneration

  • robust: chop into head, mid, tail, grows three flatworms

• negligibly senescent, self-renewing, pluripot SCs

  • low senescence → don’t easily enter permanent cell-cycle arrest

• post-mitosis, senescing somatic cells

Neoblast cell type = adult somatic SCs in flatworms

cut → neoblasts stimulated → new somatic cells

WT: neoblasts → development, injury: wound healing response → neoblasts fill in gaps from injury

why did evo redxns in capacity to regen happen?

• mammals no neoblasts, how to stim. somatic cells to regen like planaria?

• model organism: vertebrate model like axo, zfish closer in evo than flatworm (anatomy, genetics)

  • lizard tail regen, bird cellular regen of hearing

• theory: species complexity incr., heart regeneration potential dcr.

  • heart regen: zfish all, Ms within 7 days postnatal, pig 2 days postnatal, human little

3 fundamental questions

1. why might regeneration be lost?

   1. differences/similarities between high R species and low to none R mammals?

   2. differences/similarities btw dev & regen programs?

      1. R = reactivation of dev, but still somewhat distinct from dev

how axo regenerates

• can regenerate a lot: lung, liver, limbs, tails

• LD vs LR

  • axo regen mostly recapitulates emb. dev

  • blastema reactivates developmental programs

  • dev. and R pathways overlap + distinctions (FSTC1, CTRC1 genes only in R)

  • emb. limb bud in adult context

Limb Bud and R Similarities (3)

1. same pathways: fgf → growth (AEC, AER), shh → patterning (limb bud ZPA, blastema gradient), wnt → DV id, hox → positional id

2. same structural logic: prolif with undiff. cell types, AER vs AEC

3. Same Patterning Strat: gradients specify AP PD axes, feedback loops coordinate growth+patterning

Limb Bud and R Differences (5)

1. cell source: emb prog cells vs. dediff adult cells with lineage restriction + retention of positional memory

2. starting context / environment: embryo vs injury/wound (ECM remodeling + immune response) emb vs injury context

3. Structure: AER + ZPA vs AEC + blastema, AER similar to AEC / fgf signaling

4. Nerve Dependence: required in LR for growth signals // not req. in dev

5. Growth Control: dev. has time points when BMP-|AER fgf, regen has feedback loops intx w/ nerve → match existing scale/prop

Summary: starting point, cell source, structure, signals, context, growth control, outcome

bud vs blastema

emb prog vs dediff adult cells

AER ZPA AEC blastma

signals: fgf shh wnt hox

emb vs injury

dev timing vs nerve dependent + scaling

build limb vs rebuild limb

same genes in LD, LR how to test separately?

signaling: fgf shh wnt hox

KO FGF gene from birth → lethality / emb dies / fgf important → can’t study regen

use Conditional MT : controlling when/where gene is active

• normal during dev.,

• turned off later or just in specific tissue

• Conditional KO: CreLoxP → CreER + TAM injection temp control

EXP: conditional KO fgf signaling in adult tissue only after limb dev. completed then amputate limb (3)

1. limb regeneration impacted or fails

2. AEC secretes fgf but fgf signaling KOed (ie receptor cant respond to fgf)

3. blastema failed to prolif

how to use same genes in LD AND LR?

• enhancers!!!

• enhancer —- regen enhancer —— downstream fgf gene

• region includes ± 50 kB up and downstream of fgf gene

  • DEV cues act on enhancer

  • INJ cues act on enhancer and regeneration enhancer for regen.

• Regeneration programs associated with enhancer regulatory regions

  • regen enhancer for regen context

  • injury context so chromatin accessibility incr → sites open → TF bind → fgf genes activated

identify regen-regulating regions? id enhancers, transcription, TFs

uninjured vs injured state, same cell

ATACseq to assay chromatin accessibility (tied or loos in nucleosomes)

same species, diff size i.e chihuahua vs great dane

• dog is because hormonal control of growth duration: IGF1 (insulin-like growth factor) allelic differences, expressed in 8 cell blastula → death

controlling growth feature: allometry

• allometry = GROWTH IS COORDINATED

  • within organs (what cells become what share, organ itself)

  • between organs and tissues (proportion)

    • due to hormones

    • i.e end of pub: sex hormone spike → feedback loop → growth hormone shut off

Theoretical Ways to Control Organ Size (3)

• number of cells (cell div vs death)

• size of cells

• accretion: accumulation of ECM

allometry how can growth/prolif of individual cells dispersed throughout tissue/body be collectively regulated to produce organs of stereotype size? + EVIDENCE for extrinsic, intrinsic

extrinsic: functional feedback = extrinsic input into organ size

EVIDENCE for functional feedback controls size

• 2 frogs duplicated body axis connected by circulatory system → remove one liver. outcome: remaining liver is 2X as big

• spleen primordia dispersed, when collective = normal spleen size

not just dependent on organizer/patterning center, can adjust

intrinsic: intrinsic tissue control

• limb bud i.e transplant limb bud → limb bud growth to size of donor not host

  • small species → big, quail limb bud on chick, quail AER/quail fgf signal amt → quail size

• even though the liver size is under fxnal/extrinsic feedback, there’s different cell types that are under intrinsic control of cell type/proportion

liver regeneration potential reasoning

• liver processes toxins, skin withstands assault → more flexible R potential

Drosophila Imaginal Discs / Growth Contr

• pupa → fly (worm → thing w eyes, legs, antenna)

• pupa contains ImDiscs → unfurl when moulting into fly

• can culture ImDiscs (they are hearty) and expose them to moulting hormone → activate adult dev. program

Drosophila ImDiscs EVIDENCE for Intrinsic Growth Control (4)

1. Disc transplantation between species: donor disc becomes donor size

2. limit # of progenitor cells

   1. laser ablate some progen. cells → get normal size ImDisc

3. kill progenitors & stop cell div.

   1. ImDisc → kill ALL BUT 4 CELLS → get normal size ImDisc with 4 humongous cells (didn’t divide but grew in size)

4. Mosaics with cells of different sizes growing @ different rates

   1. small minute cells divide slower than WT

   2. mosaic of minute/WT → WT cells slow cell div and wait til min cells catch up

   3. bc min destined to take up a certain size, so WT not gonna take up excess

Conclusions: growth control program is intrinsic to ImDiscs

• it will always grow to the size it is supposed to, intrinsic to the species

program is flexible: alter it a lot → still get normal ImDisc size

mosaic: cell-cell communication is occuring

individual cell decision (divide or not) based on communication with other cells

Summary of Growth Control (4)

1. organ size controlled by intrinsic dev. programs w/in organs, between organs/tissues

2. some intrinsic programs more flexible, can have fxnal feedback

   1. we don’t know if signals are circulatory factors extrinsic to organ or functional feedback is intrinsic to organ, or both

3. size limited by progenitor pool (i.e panc vs liver)

4. ultimately size associated with # cells produced, size of cells

EXP: ImDisc with hippo MT

EXP: liver with hippo MT

1. ID bigger than WT

2. liver bigger than WT

3. rescue the size by expressing hippo

EXP: Screen: is cell taking up more or less of fair share of space?

• early in dev, ake cell thats heterozygous MT

• use Flp/frt recombinase that results in cell div: one homozygous WT, one homozygous MT

• mark 1 cell type with GFP

• see if it takes up more or less of organ share

  • 3 outcomes: blue xs vs. WT, WT same size, blue less vs. WT

MR that affects ID: Worts + EXP

• warts MT takes up excess of fair share compared to WT

• express wts MT on ImDisc that becomes leg → out of control growth on leg

HIPPO SIGNALING PATHWAY

• sav cofactor bind to hippo kinase

• LATS gets phosphorylated

• p-LATS phosphorylates YAP/TAZ

  • YAP/TAZ normally goes to nuc + coactivator for TFs (not directly binding DNA)

  • p-YAP/TAZ → sequestered or degraded/ub

• HIGHLY CONSERVED molecule + function

  • yeast, Dmel, mammals → some common ancestor had hippo signaling

YAP/TAZ controls Important Genes (4) + (1)

• cell cytoskel, cell jxn genes

• cell cycle genes / YAP/TAZ promote cell division (cancer)

• cell EMT & migr / slug, snail (cancer)

• cell plasticity/polarity / MYC OCT4 SLX2 NANOG Yamanaka factors for iPSCs

  • dediff. into SC-like state

YAP/TAZ doesn’t activate ALL genes at once

Outcome depends on co-TFs that it binds

• hx matters: coTF abc vs co-TF acd

EXP: induce heart damage in Ms, add active YAP/TAZ →

outcome: heart regeneration

since YATA associated with dediff into SC-like state

requires PRECISION: too much YATA → too much heart regrowth → Ms death

allow growth then stop growth

Inputs to Hippo Pathway (upstream) Regulate YAP/TAZ

• master regulator receives inputs:

  • cell pol./adh., mechanical cues, cellular stress, extracellular signals, mechanical tension

• acts upstream of YATA

  • hippo controls YATA, YATA executes txn

• cell culture: mechanical tension at confluence → input into hippo → hippo ON YATA inhibited → growth genes turned off → stop growing

what causes aging

• telomere shortening

• accum of DNA dmg

• SCs decrease, SC dmg

• reactive oxidative species

• diminished Unfolded Prot Response

• genetic program that advances life stage

  • similar to how apoptosis is programed cell death → programmed aging

theory based on EXP: caloric restriction

calorically restricted Ms without malnutrition → longer life

less food → less metabolism → less toxic bypdts of metabolism → healthier live longer, delayed age related disease

healthspan vs lifespan

• lifespan: live forever but in bed is not ideal

• extend healthspan: live to 100 drop dead after / good functional health more important

CR theory

EXP: related to oxidative dmg? →

EXP: related to metabolic rate? →

NO. reduced oxidative dmg → no change in lifespan

yes. correlated, lifespan incr, metabolic rate dcr inverse relo

• big vs small dogs HOWEVER MANY EXCEPTIONS same size → but 23 vs 3 yrs rat squirrel

EXP EV for: Aging is under genetic control!!

MT in C. elegans

• isolated 2 MTs

• age1 MT → 2x lifespan

• daf2 MT → 2x lifespan (dauer defective2)

proof that aging is under genetic control

C. elegans life: emb → L1 thru 4, emb → can enter Dauer/Diapause State, aka times are bad → hibernation / dcr metabolism incr innate immunity

30 different dauer genes

• go into constitutive Dauer state

• prevent going into Dauer state

daf2 complete LOF → constitutive Dauer state (live forever BUT stuck in diapause)

constitutive aka stuck

EXP: daf2 temperature sensitive allele or RNAi →

control function of dauer

permissive temp (cold): WT daf2

non-permissive temp (dysfunctional): temp shift in any stage L123 → constitutive Dauer

temp shift in later L4 → 2x lifespan

timing of MT matters

EXP: daf2 and downstream daf16

daf2- (daf16 wt) → 2x lifespan

daf2- daf16 - → normal lifespan

• so life extension from daf2 MT REQUIRES FXNAL DAF16

what is daf2 and daf16?

• daf2 is an insulin receptor

  • DAF2 is CONSERVED IN mammals (1 insulin + 3 insuline-like GF, 5 inR → IGF in hormone size variation), drosophila (7,1), C. elegans (40 insulin peptides, bind to 1 daf2 inR)

• daf16 is in foxO T.F. family

Insulin Like Signaling Pathway (IIS) and CR Condition

og: insulin receptor → kinase cascade → p-FOXO → can’t enter nucleus/inhibited

• daf2-: no insulin receptor → FOXO enter nuc → longer life genes (?)

• daf2-daf16-: no IR, no FOXO → reg life

food → IIS (IR on, FOXO phosphorylated) → FoxO phosphorylated/inhibited → Foxo down

caloric restriction → IIS decreased signaling → FOXO not phosphorylated/active → Foxo up

MORE Central Regulation (than IIS) : TOR (target of rapamycin) + tor signaling

• inhibition of TOR increases lifespan in all species investigated (2x life in C. elegans, Ms, flies, yeast)

• member of Ser/Thr kinase family, not receptor

• MASTER REGULATOR

  • sense nutrients, hormones, GFs

  • TOR encompasses many pathways including IIS

• energy high → TOR ON → fast living/aging → growth, development, reproduction

• energy low → TOR OFF → rest, repair (wait until growth reprod available again)

2 TOR Complexes

• not individual prot, complex

• both receive food/nutrient signals (energy high or low)

• insulin IIS pathway requires TOR too

• TORC1

  • more sensitive to rapamycin

  • rapamycin -| TORC1

  • free or tethered to lysosome

• TORC 2

  • less sensitive to rap: high [rap] long duration -| TORC2

Other Signals Mediate Lifespan

• besides insulin, TOR

• also: SIRT AMPK

• more complicated

Summary of Aging Contr

• life stage advancement (aging) is under genetic control

• restricting food → longer life

• restricting food in TOR MT, or IR MT → not longer life

  • Input: CR → TOR → IR → longer life

  • MT breaks input → output, TOR/IR mediate this

  • tor + IIS

Dietary Restriction vs CR

• caloric restriction increases health

  • sufficeint nutrients, low calories (less food, same nutrition)

  • but u need to maintain full nutrition / eat ONLY powders

• dietary restriction: minimal nutrients, normal calories (same calories different composition, change nutrition/specific nutr dcr)

  • found dietary restriction where 2x life and still healthy

DR EXP:

DR: minimal nutrients, normal calories → 2x life and stilly healthy

manipulate diet composition, carb/prot ratio

high carb, low protein → healthier

high prot, low carb → less healthy

in Ms experiment: hi-c low-p 4X better than hi-p low-c

Tradeoffs on Aging Control

• treatments that extend lifespan but not necessarily healthspan

• tradeoff between lifespan and fertilization

  • if you eat rapamycin to slow aging → you become sterile

• timing of intervention matters

  • C. elegans dauer state induced post reproduction

  • younger ages are still growing and developing

• factors that are upregulated in aging could be beneficial not detrimental

Summary of Aging Control Lecture p1

CR not only extends life, but extends HEALTHSPAN

TOR MT, IR MT 2x life in C eleg, Ms, flies, yeast

CR in TOR MT, IR MT → not longer life so intact TOR and IR are needed for CR extend life

EXP: DR + components

DR: normal level calories, control mactronutrients, control micronutrients

establish DR diet leading to max life / / flies 2x lifespan

• this has all nutrients and extends life

tested Ms, flies, monkeys

add additional components (maintain same calories)

• DR + carbs/lipids → 2x lifespan

• DR + non-essential AAs → 2x lifespan

• DR + essential AAs → 1x lifespan (not ext)

• DR + branched chain AAs → 1x lifespan (not ext)

late in life/post reprod, high protein is bad

high prot assoc. with diabetes

AAs/Glycine and Longevity + EXP

Glycine

• cells in old worms accumulate Glycine (gly storage proportional with age)

• increasing Glycine consumption → EXT 1.3 X LIFESPAN

  • feeding glycine L1-L4 / D3 lay eggs → no change

  • feeding xs glycine to old worms post day 5 → 1.3X life

AAs/MET and Longevity + EXP

Methionine / essential AA (worms, flies, Ms)

• normal diet + limit Met → longer life

• normal diet + excess Met → shorter life than avg

DR regime - minimal diet, full nutrients that ext lifespan

DR + essential AAs → 1X life (avg/not ext)

DR + essential AAs - MET (minimal met) → life was extended

so excess Met shortens healthspan?

DR + MET only → still extends lifespan

• test different conc

• at lower level, incr, higher level, dcr

ratio of AA matters

Darwin’s Demon doesn’t exist / tradeoff between ___ and ____

• no organism born reproducing already, reproduce max progeny/unit time and live forever

• not possible to reproduce at a young age

• energy is finite/limiting

• trade off between:

  • somatic repair, germline maintenance

  • growth and reprod

scarce times, no food, no mates → somatic repair, germline maintenance

good times, food, mate access → growth + reproduction + cost of aging

DR, IR-, TOR- → LIFESPAN incr but FECUNDITY dcr / fewer progeny

EXP: germline ablation →

EXP: exception

germline ablation/sterilization → fewer progeny (0) and 2X lifespan

DR + MET → still 100% fecund & 1.4X lifespan

Other Theory: Antagonistic Pleiotropy

• if aging under natsel, why allow aging?

• selection acts to optimize reproduction to maintain early health

  • alleles required for higher reproduction cause aging later in life

  • cancer: cell div when young, cancer when old

Global Regulation of IIS and TOR EXPs (neurons, fat storage)

• IR-FoxO wt → 2x lifespan

• IR- FoxO- → 1X lifespan

• IR- FoxO- + FoxO wt in neurons → 2X lifespan

  • FoxO required in neurons

• ablate sensory neurons / odorant receptor MT → extend lifespan (Ms eat food but can’t smell it/can’t sense energy)

• fat storage: intestine of worm, fat body of flies →

  • remove fat body + CR → no ext life

  • remove fat body IR MT + CR → no ext life

    • energy state neurons sense energy state → communicate thru fat storage organs → communicated to cells throughout body / central reg

Summary Cont Aging P2

• IIS, TOR, AMPK, SIRT important for control aging

• DR most effective in lengthening lifespan

  • timing of DR matters: post reprod

  • DR effects work via different signaling pathways

• cells don’t individually assess nutrient state of entire organism // organism sense nutrient state and communicate to cells → CENTRAL REG of this program (fat bodies)

• neuron system essential for lifespan lengthening (only need Foxo/Daf16 in neurons)

• can u extend healthspan w no consequences

• microbiome?

evolution and development are related

• correlation

• development in evo context (conserved vs not conserved)

  • we learn human dev. from worms, Ms, flies, yeast bc conserved pathways

evolution to humans / ancestor →

1. animal life developed multicellularity (developed independently 6x)

   1. sponge no germ layers

2. developed germ layers 3

   1. comb jelly only has 2 germ layers: ecto (skin CNS), endo (organs) NO MESO (muscle)

3. how ga. works: deuterostome (vs protostome)

deuterostome: chordates/echnoderm aka. human ga.

• blastopore goes in/ga. start = anus, ga. end = mouth

protostome: invertebrate/worm flies, snails ga.

• cells still invaginate, secrete bmp inhibitors at blastopore/Org

• ga. start = mouth, ga. end = anus

• swapped DV axis

urbilateria

metazoa

bilateral symmetry

all multicellular animals

how did diversity of animal life evolve? Cambrian Explosion

600 mya: earliest urbilaterian, nothing

545 mya: no representatives of any of these phyla

540 mya: representatives of all phyla

• use fossils for representatives but not accurate with time / squishies have no fossils, use shell fossils

FAST

• in 60 million years dev. all phyla, vs.

• it takes 75-100 million yrs for comon ancestor to speciate into humans and rodents (in one phyla chordates)

• took 20 million years for common ancestor to speciate into rats and mice

what feature of the ancestor of bilateria do all animals have?

• genetic toolkit

• modular organization

assemble GT + have modules → EXP w/ them = diversity

i.e signaling pathway is part of genetic toolkit

• all phyla including sponge, jelly fish

• if even sponge and jelly have HOX gene (like how humans do) → last urbilateria common ancestor must have HOX gene

Genetic Toolkit in Ancestor of ALL Animals

• signaling pathways

  • TGF-b, wnt, notch, shh, RTK

  • evidence at LCA, before evolution → all present

• selector genes (master control genes)

  • pioneer TFs

  • influence fate of many cells (tissue dev, structure)

  • active in different locations

Selector Genes Realm of Action / location

• i.e hox gene: region specific along A-P

• twist: tissue specific

• mef: make muscle, cell type specific

• Engrailed: segmentation, at posterior of each segement (A-P in segment)

• Eyeless, Vestigial: TFs make eye, make wing

Master Control Gene Pax 6

• eye development + evo insights

  • flies compound eyes, octopus different eyes, etc. → og thought it was convergent evolution, but its not

• babies, Ms without eyes vs. eyeless flies

• cloned human eyelessness: its Pax6 (paired homeodomain), ortholog of Ey in flies

• very different eyes: photoreceptors early on, later on: changes in eye

EXP: misexpress Pax6 on wing/antenae/leg→

EXP: can use human pax6 in flies, rescue Ms pax6 MT with Eyeless gene from flies

get whole ye on leg

considered a master control gene bc it executes whole eye development program

mice Pax6 MT + flies Eye gene = rescued eyes

• Pax2 so conserved it can functionally compensate across different species

Modular Organization

• how to get from no animals to a lot in a short period

• same basic genetic toolkit

• MO is something evolution can act on independently

• it’s a development subunit that natural selection can act on one module without affecting anything else

  • ie changing roof motor of car only, and car still works

• embryonic structure showing developmental morphological individual

• elements are correlated with adult structure (limb bud becomes limb)

Module Organization Example: Limb Bud

• homologous structures in limb bud across human, mole, horse, dolphin, bat

• general same but modular changes to specific parts: moles have extra wristbone, horse phalange is hoof, dolphin knuckle duplication, bat super long phalange

• evolution independently experiments on parts of limb bud

• evolution independently experiments on fore vs hindlimbs i.e bird (legs and wings are diff)

HOX Genes on diversity

• responsible for AP identity

1. duplications and deletions are important

   1. flies vs humans → 1 vs 4 sets of Hox

2. control regions are important (small, tinkerable)

in the 4 hox clusters, p1c1 p1c2 p1c4 are orthologs with fly p1

p1c1 p1c2 p1c4 are paralogs with eo inside human

• paralog fxn depends on natsel EXP

• multiple domains, combining domains

Ortholog vs Paralog

• orthologs arise from speciation

  • species 1 has gene X

  • over time, at some point, two groups stop interbreeding/become unable too → speciation of species 2 and 3 both have gene X

• i.e Pax6 humans and Ey flies are gene X, orthologs of each other

• paralogs

  • over time, gene X is duplicated → gene X’ and gene X: gene X’ either becomes evo dead end or natsel experiments with it and it can adopt 2nd fxn

ortholog

👉 Same gene in different species

• Same “job,” different organisms

paralog

👉 Duplicated genes within the same species

• arise by gene duplication

• can evolve new or specialized functions

Change in Gene Expression Creates Morphological Diversity i.e HOX

• change in expression domain → major change in body organization

  • express gene in diff place → eye on leg (same expr, diff place)

• change in regulation of Hox genes is → diversity of # and identity of repeated modular units

how did snakes lose their legs

EXP: replace Ms hoxc6 with snake hoxc6 → what happens

• HOX expression in chick, combo of hoxb5 c8 c6 → forelimb dev in front of it/combination

• snake has hox b5c8c6 all the way to the top, near bottom: occasionally sm snake species i.e python get tiny hindlimb bud, but it absorbs it later

EXP: get normal Ms dev

• its not the hoxc6/protein itself

• it’s the control region

how do hox domains shift during evolution?

the regions of the body where specific Hox genes are expressed change over evolutionary time, leading to different body plans.

• modular protein that regulates hox gene expression

  • change TFs that bind hox DNA

• modular cis-regulatory region of hox genes

  • modify regulatory region of hox genes

  • cis-reg element / the entire DNA region that regulates a gene’s expression i.e enhancer

    • regulated by minimum of 4-6 TFs

    • combinatorial action to inhibit activate

    • topology matters → get a big change from changing 1 nt

Co-evolution of Cis-Reg Elements

• enhancer region controlling hoxc6

• ortholog in Ms, whale, chick → 5 different regions where TFs can bind

• TF binds as a dimer, recognize 6-7 nts (1 dimer recognize/intx with 3 nt)

• within the entire Ms genome, you can change 1 nucleotide within control region (cis-regulatory element) → changes where hoxc6 expressed → changes limb location

EXP: chick hoxc8 enhancer drives Ms hoxc8 gene →

you get hoxc8 protein expressed, but its pattern of expression looks like a chick → shifts where forelimbs form

exact location where limb forms is different

role of regulatory DNA / CRE

• modularity of cis-reg element: individual element is involved independently

• TF combinatorial action → small change, different expression pattern

5 finger formation, no more than 5 in animals

• shh, hox genes → 5 digits

• 5 is the max because only have 5 hox genes expressed in limb

• evolution history constrains development

  • hox9, then hox910, etc

• you can have more than 5 digits BUT there are not more than 5 unique digits

  • >5 toes,

  • AER duplication in feet → mirror image toes