Regulation of germline stem cell proliferation downstream of nutrient sensing

Review

Introduction
 Stem cells proliferate in two ways
o Asymmetrical – two different daughter cells, one resembles mother (stem) cell
and other committed to differentiated fate
o Symmetrical – two identical daughter cells that are same as mother cell
 Short-ranged signals from niche cells (DTCs in C. elegans) prevents nearby GSCs from
differentiating; these signals activate molecular cascade within GSCs that alter gene
expression to specify and maintain GSC identity
 Signaling from niche cells determine size and rate at which GSC population proliferates,
depending on level at which it regulates GSC identity
 Timing of stem cell division may be regulated by microRNA-dependent down regulation
of Decapo, a p21/p27 cyclin-dependent kinase (CDK) inhibitor, thereby relaxing controls
on G1/S transition
 When environmental conditions unfavorable, rate of organism development delayed
and this response includes insulin, AMPK, an TOR signaling pathways
 GSCs have important genetic information so must be guarded from deleterious
mutations
o Transposons silencing mechanisms are more efficient in germ line compared to
soma
o Cell cycle quiescence to minimize risk of deleterious mutations due to cell
division during periods of insufficient energy

Nutrient stress blocks stem cell divisions

 GSC divisions delayed when nutrient deprivation
 Eg. When cholesterol levels insufficient, brood size of C. elegans are reduced due to
defect in germline proliferation and differentiation
 GSCs must sense nutrient quality and abundance or they must gauge general metabolic
status of animal to adjust division accordingly

Insulin signaling regulates the rate of GSC divisions

 Poor-environmental conditions during early post-embryonic life, including limited
nutritional resources and high population density triggers entry into dauer
 Disrupting function of positive components genes of insulin-like cascade (eg. Insulin-like
growth factor (IGF) receptor orthologs (daf-2) catalytic subunit of PtdIns3-kinase (age-1),
PtdInsP3-dependent kinase (pdk-1), or Akt/PKB (akt-1/2)) results in down regulation of
metabolic rate and induces constitutive dauer arrest
 Disrupting function of negative antagonistic components genes of insulin-like cascade
(PtdIns-3 phosphatase PTEN (daf-18), FOXO-like forkhead transcription factor (daf-16))
disrupts ability of animals to enter dauer
  Reducing insulin-like signaling later in life after window of dauer development, inhibits
gamete production dramatically

Nutrient regulate GSC division rate through an insulin-dependent neuro-endocrine signal

 C. elegans have several insulin-like peptides that are mainly expressed in neurons and
either positively or negatively regulate activity of a unique insulin/IGF- receptor
homolog
 Experiments designed to restore function of insulin-like receptor (daf-2), PtdIns3-kinase
(age-1), or downstream target of pathway the FOXO-like forkhead transcription factor
(daf-16) have shown that daf-2 and age-1 activity in neurons sufficient to sustain
reproductive development in daf-2 or age-1 mutant animals
 Unclear whether neuronal daf-16 activity is sufficient to couple GSC proliferation with
reduced insulin-like signaling
o Hypothesis may be increased insulin-signaling influence production of sterol-
derived hormone by cytochrome P450 DAF-9 n hypodermis or/and pair of
neuroendocrine cells
o Hormone may affect nuclear hormone receptor called DAF-12, promoting
reproductive development
o Daf-2; daf-16 double mutants cannot execute dauer or block GSC divisions, but
still grow into sterile adults, indicating insulin-like signals regulate GSC division
and germline development through daf-12 independent mechanism

Insulin levels do not seem to affect niche-GSC signaling

 Unclear whether insulin-like signaling affects manner with which niche communicates
with GSCs
 Delta/serrate-like ligand called LAG-2 expressed by DTC activates Notch receptor in GSCs
promoting proliferation and does not seem to be affected by changes in insulin-like
signaling
 Possible that the notch receptor does not promote proliferation but rather specification
of GSC identity, which is prone to be proliferation

PtdIns3-phosphatase PTEN regulates GSC divisions in a FOXO-independent manner

 PTEN acts downstream of insulin-like receptors, counteracting PtdIns3-kinase
o Loss of PTEN results in an increase in PtdInsP3
o Elevated PtdInsP3 levels activate a complex that includes Akt/PKB in a PDK1-
dependent manner, which phosphorylates and inhibits FOXO forkhead
transcription factor from entering nucleus
 Found that daf-18/PTEN-dependent; daf-16/FOXO-independent regulation of GSC
divisions occur
 C. elegan hatchlings do not being post-embryonic development and two initial GSCs do
not proliferate until animal starts feeding
  Quiescence of GSCs in starved L1 larvae requires activity of PTEN and inappropriate
divisions that occur in daf-18 mutants are suppressed by mutations in age-1/PtdIns3-
kinase or akt-1/Akt/PKB
 Daf-16/FOXO null mutation does not bypass food requirement for GSC proliferation
 PTEN required to downregulate GSCs proliferation but FOXO is almost not needed in
most conditions
 Reduced insulin-like signaling downregulates GSC proliferation partly through FOXO-
independent PTEN and/or Akt/PKB targets

Mysterious G2/M arrest of low insulin-induced quiescent GSCs

 Insulin signaling affect cell cycle machinery by regulating Akt/PKB activity which is
required for progression through G1/S and G2/M checkpoints
 Adult stem cell division regulated by two G1/S regulators p21 and p27, which is
regulated by Akt/PKB
 GSC quiescence under nutrient stress occurs at G1/S checkpoint through inhibition of
Akt/PKB-dependent p21/p27 downregulation
 RNAi depletion of p21/p27 orthologues in C. elegans induce GSC hyperproliferation
during dauer formation but not during early reproduction development when nutrients
are not limited
o Implies that p21/p27 orthologues control GSC division in nutrient deprivation
o GSC do not divide even when p21/p27 CDK inhibitor homologs are depleted in L1
starved larvae
 Suggests that GSC does not arrest in G1 but at G/M checkpoint in starved/insulin-like
compromised larvae
o GSCs of starved L1 animals have replicated DNA content with condensed
chromosomes and duplicated centrosomes (S-phase)
 Indicates that insulin-regulated quiescence of GSCs occur at G2/M checkpoint and G1-
specific CDK inhibitors may contribute to deceleration of GSC divisions associated with
stress

The LKB1/AMPK cascade links GSC division rate with insulin levels
 Aak-2 is a downstream effector that links GSC proliferation rate with insulin-like
signaling levels
o RNAi of aak-1 (alpha1 catalytic subunit) gives phenotype similar to aak-2(RNAi)
o Inactivation of both subunits results in pronounced hyperplasia of germline,
suggesting additive function
 AMPK activated allosterically by AMP and requires key activating phosphorylation at
very conserved site to be fully catalytically activate
o Major AMPK-activating kinase was identified as LKB1/SK11 (tumour suppressor)
o Inactivation of LKB1 homolog (par-4) causes germline hyperplasia in dauer with
severity similar to aak-1/aak-2 double mutants
o Very highly conserved LKB1-AMPK cascade in insulin-dependent regulation of
GSC division rate
 Akt/PKB and AMPK linked to regulation of mTOR growth pathway
o Akt/PKB and AMPK antagonistically regulate activity of TSC1-TSC2 complex
(human tumour suppressor) through direct phosphorylation of TSC2
o When insulin signaling is elevated and AMP:ATP ratio is low, TSC complex is
antagonized by Akt/PKB and is not activated by AMPK
o Mutations in TOR orthologs cause larvae to arrest with underdeveloped germline
 Likely that TOR signaling couples GSC division rate with nutrient status in insects
 Unclear whether LKB1 contributes to regulation of GSC divisions by insulin-like signaling
levels