This document summarizes a presentation on the role of the DAF-2 receptor pathway in regulating longevity in C. elegans. Key points include: 1) The DAF-2 receptor integrates environmental signals like temperature and regulates processes involved in growth and survival. 2) Mutations in daf-2 can both positively and negatively impact longevity, growth, and reproduction depending on temperature. 3) Experiments examining expression levels of daf-2 ligands identified pro-survival, anti-survival, and condition-specific ligands. 4) Further experiments showed that inhibiting protein synthesis extends lifespan at 25°C but is harmful at 15°C, and this effect is dependent on daf

1. To live and grow:
Mechanisms of longevity via a
growth hormone pathway
Angela Sanchez
CSB
Larsen Lab

2. Goals for today: Feedback on current data
• New Data
• Critiques
• Key experiments for model testing
• HOLD QUESTIONS/COMMENTS for first portion
• Then free for all

3. daf-2 receptor integrates environmental signals
DAF-2 RECEPTOR
• Integrates environmental signals
• Originally dauer formation
• Mutations extend adult lifespan
Nutrients
Temperature
Pheromone
Growth Survival
Organismal Response

4. Main Question: Signaling of Longevity
DAF-2 RECEPTOR
LONGEVITY
Signaling that
promote survival?
Comfortably mimic effects……without negative impact

5. daf-2 mutants: Positive and Negative Effects
DAF-2 RECEPTOR
• daf-2 affects many processes
• Mutation negatively impacts
• Growth
• Reproduction
Growth Survival

6. daf-2 mutation negative impacts
% Growth to Adult % Brood Size Reduced Movement
Allele 15˚C 25.5C 15˚C 25˚C 15˚C 25.5C
WT 100 100 100 100 No No
Class I
M41 100 0 100 88 No No
Class 2
E1370 100 0 76 39 No Yes
M596 100 0 71 89 No Yes
m579 100 0 73 67 No Yes
(adapted from Gems 1998)
Capture the benefit…..avoid the risk

7. Identifying Mechanisms of Survival
1) Longevity Variable: COOL TEMPERATURE
2) Sorting: ALLELIC SERIES
Signaling that
promote survival?
identify what is benefical or detrimental

8. Cool Temperature and daf-2 mutation:Similar effects
PHENOTYPE TEMPERATURE DAF-2
Lifespan DOUBLES DOUBLES
Development DELAYED DELAYED
Oxygen
Consumption
REDUCED REDUCED
Temperature daf-2 mutation

9. DAF-2
daf-2 alleles have weak and strong
lifespan effects
m41
G380E
m579
R434C
m596
G544S
e1370
P1465S
MILD
SEVERE

10. daf-2 mutants: abnormal temperature response
0
20
40
60
80
100
0 20 40 60
daf-2(m41)
Ligand binding domain
15˚C vs 25˚C
daf-2 25˚C vs WT 25˚C daf-2 15˚C vs WT 15˚C daf-2 15˚C vs daf-2 25˚C
Wild type
15˚C25˚C

11. Promoting Survival or Growth: daf-2 Ligands
DAF-2 RECEPTOR
LONGEVITY
39 daf-2 ligands
Growth
DAF-2 LIGAND
Dauer

12. Sorting out daf-2 ligands
• Which are expressed in adult?
• Which are pro-survival?
• Which are anti-survival?
• Monitor mRNA levels 39 ligands
15˚C Wildtype PRO-SURVIVAL
25˚C Wildtype ANTI-SURVIVAL PRO-SURVIVAL
15˚C daf-2 vs WT
25˚C daf-2 vs WT
PRO-SURVIVAL
PRO-SURVIVAL ANTI-SURVIVAL
ANTI-SURVIVAL
Possible Function of These Peptide Growth Factors

13. Sorting out daf-2 ligands
• Day 1 adults
• Larval development at 15˚C
• 24 hours at 15˚C or 25˚C
• 3-4 Biological replicate
• qRT-PCR duplicates
– All 39 detectable
– 1000 fold range
15˚C 25˚C
Wildtype Wildtype
M41 M41
E1370 E1370
M596 M596
m579 m579

14. Environment
Effects
Gene Allele Effects
Gene X
Environment
Interaction Effects
• Environment Effects
• Gene x Environment Interaction Effects
• Gene Allele Effects
daf-2 ligands qRT-PCR Results:
Questions we will answer

15. • Wild type 15˚C vs Wild type 25˚C Up at 15˚C
Up at 25˚C
daf-2 ligands qRT-PCR Results:
Environment Effects

16. • Wild type 15˚C vs Wild type 25˚C
• daf-2 15˚C vs daf-2 25˚C
m41
e1370
m596
m579
Up at 15˚C
Up at 25˚C
daf-2 ligands qRT-PCR Results:
Gene x Environment Interaction Effects
Increasing Lifespan

17. • Wild type 15˚C vs Wild type 25˚C
• daf-2 15˚C vs daf-2 25˚C
m41
e1370
m596
m579
• daf-2 25˚C vs wild type 25˚C
• daf-2 15˚C vs wild type 15˚C
Up at 15˚C
Up at 25˚C
Higher than WT
Lower than WT
daf-2 ligands qRT-PCR Results:
Gene Allele Effects
Increasing Lifespan
Genotype Changes

18. Pro-survival Insulins
WT 15˚C vs
WT 25˚C
daf-2 15˚C vs
daf-2 25˚C
15˚C
25˚C
daf-2
daf-2
daf-2 25˚C vs
WT 25˚C
daf-2 15˚C vs
WT 15˚C

19. Anti-survival Insulins
WT 15˚C vs
WT 25˚C
daf-2 15˚C vs
daf-2 25˚C
daf-2 25˚C vs
WT 25˚C
daf-2 15˚C vs
WT 15˚C
15˚C
25˚C
daf-2
daf-2

20. Condition-specific Insulins
WT 15˚C vs
WT 25˚C
daf-2 15˚C vs
daf-2 25˚C
daf-2 25˚C vs
WT 25˚C
daf-2 15˚C vs
WT 15˚C
15˚C
25˚C
daf-2
daf-2

21. Insulin Ligand Summary
PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC
TEMPERATURE
REGULATED
6
daf-28, ins-28, ins-
29, ins-35, ins-25,
ins-20
6
ins-19, ins-11, ins-16,
ins-27, ins-34, ins-5
5
ins-30, ins-24, ins-22, ins-23,
ins-4
DAF-2
REGULATED
4
ins-33, ins-6, ins-37,
ins-21
9
ins-7, ins-26, ins-34,
ins-5, ins-2, ins-8, ins-
3, ins-12, ins-36, ins-
18, ins-15
2
ins-9, ins-10

22. Testing anti-survival ins genes
• Inverse relationship between growth and survival
• Growth assay
• Lifespan
DAF-2 RECEPTOR
Growth Survival

23. Insulin Ligand Summary
PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC
TEMPERATURE
REGULATED
6
daf-28, ins-28, ins-
29, ins-35, ins-25,
ins-20
6
ins-19, ins-11, ins-16,
ins-27, ins-34, ins-5
5
ins-30, ins-24, ins-22, ins-23,
ins-4
DAF-2
REGULATED
4
ins-33, ins-6, ins-37,
ins-21
9
ins-7, ins-26, ins-34, ins-
5, ins-2, ins-8, ins-3, ins-
12, ins-36, ins-18, ins-15
2
ins-9, ins-10

24. RNAi of ins genes: Growth Assay
• Adult hermaphrodite 24 hrs on RNAi plates
• 2 hr egg lay to synchronize
• Animals monitored for growth effects

25. ins-7 RNAi effects growth
• ins-7
– 100% embryonic lethality at 15˚C
– Delayed variable growth at 25˚C
• Results in death as early larvae
• or dauer like arrest maybe death
• ins-7 unique cellular function
• Other explanations

26. Insulin Ligand Summary
PRO-SURVIVAL ANTI-SURVIVAL CONDITION-SPECIFIC
TEMPERATURE
REGULATED
6
daf-28, ins-28, ins-
29, ins-35, ins-25,
ins-20
6
ins-19, ins-11, ins-16,
ins-27, ins-34, ins-5
5
ins-30, ins-24, ins-22, ins-23,
ins-4
DAF-2
REGULATED
4
ins-33, ins-6, ins-37,
ins-21
9
ins-7, ins-26, ins-34,
ins-5, ins-2, ins-8, ins-
3, ins-12, ins-36, ins-
18, ins-15
2
ins-9, ins-10

27. Testing anti-survival ins-19 and ins-7
Lifespan Analysis
• ins-17 RNAi
– no significant change
• ins-19 RNAi
– not significant at 15˚C
– Significant at 25˚C
• Trial one longer
• Trial two shorter
• ins-7 RNAi
– Not significant at either 15˚C or 25˚C
– Unlike Murphy and Kenyon publications

28. daf-2 receptor levels?
Reduced by warm temperature in longest lived allele

29. Conclusions for discussion
• All ins genes are made
– with wide range of expression levels
– Competition for the receptor occupancy
• ins gene expression is controlled by multiple factors
• ins gene functions may differ in development and
adult longevity
• Long life achieved by
– enhancing survival patterns
– reducing life limiting factors
– RISKY

30. Main Questions: Signaling and Mechanisms of Longevity
DAF-2 RECEPTOR
LONGEVITY
Signaling that
promote survival?
Mechanisms of longevity?

31. Microarray: 25˚C vs 15˚C
mRNA mRNA
Compare
PROTEIN
SYNTHESIS
LYSOSOMALMETABOLISM

32. • eIF4 G
– Translation Initiation Factor
– Inhibition Increases lifespan
– Repressed at 25
• eIF3 A
– Translation Initiation Factor
Temperature Regulated Translation Genes
Environment
Effects
Gene Allele Effects
Gene X
Environment
Interaction Effects

33. Temperature Regulates Protein
Synthesis
Is protein synthesis required for longevity at 15˚C?
Wild type?
daf-2 mutants?

34. 0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
daf-2(m41)
daf-2(m577)
daf-2(m596)
daf-2(m579)
daf-2(e1370)
p= 0.2576 p= 0.9445
p= 0.938
p= 0.619
p= 0.3825
p= 0.0003
wild type
(Takano, Sanchez, Larsen, unpublished)
15°C eif4G RNAi

35. • Inhibition does not increase lifespan in wild type
• Inhibition results in sickly/paralysis phenotype
• Required for lifespan extension in
– m579
– e1370
eIF4 RNAi harmful at 15˚C

36. Temperature Regulates Protein
Synthesis
Is protein synthesis decrease
pro-survival or anti-survival at 25˚C?
In wild type?
In the daf-2 mutant background?

37. 0
20
40
60
80
100
0 10 20 30 40 50
0
20
40
60
80
100
0 10 20 30 40 50
0
20
40
60
80
100
0 10 20 30 40 50
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m41)
daf-2(m577)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m596)
daf-2(m579)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(e1370)
25°C eif4G RNAi
p<.0001 p= 0.6831
p= .0014
p= 0.4785
p= <.0001
p= 0.0012
wild type
(Takano, Sanchez, Larsen, unpublished)

38. • Inhibition increases lifespan in wildtype
• Effect enhanced in daf-2 mutants
– Late in life liability
eIF4 RNAi extends lifespan at 25˚C

39. 0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
0
20
40
60
80
100
0 10 20 30 40 50 60
daf-2(m41)
daf-2(m577)
daf-2(m596)
daf-2(m579)
daf-2(e1370)
15°C eif3A RNAi
p= 0.0799 p= .4760
p= .0034
p= 0.2573
p= 0.0694
p= 0.838
wild type
(Takano, Sanchez, Larsen, unpublished)

40. • Inhibition does not increase lifespan in wildtype
• Inhibition results in paralysis/hernia phenotype
• Required for lifespan extension in
– m579
– m41
eIF3 RNAi harmful at 15˚C

41. 0
20
40
60
80
100
0 10 20 30 40 50
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m41)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m577)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m596)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(m579)
0
20
40
60
80
100
0 10 20 30 40 50
daf-2(e1370)
25°C eif3a RNAi
(Takano, Sanchez, Larsen, unpublished)
p<.0001 p<.0001
p<.0001
p<.0001
p<.0001
p<.0001
wild type

42. • Inhibition increases lifespan in wildtype
• Effect enhanced in daf-2 mutants
eIF3 RNAi extends lifespan at 25˚C

43. • Effect is CONDITION and GENOTYPE specific
• Translation inhibition is harmful at 15˚C
• Effect ENHANCED in daf-2 mutants
• Not additive
• daf-2 mutation overcomes life-limiting barrier
Conclusion: Be precise about context

44. Why is lifespan extension enhanced in daf-2 mutants?
PROTEIN
SYNTHESIS
LYSOSOMALMETABOLISM
DAF-2 RECEPTOR
Temperature

46. A long long time ago……….. (1981)
in a lab far far away……… (missouri)
mutagenesis screen for dauer formation

47. daf-2 allelic variation
daf-2
cxTi9878 e1369 m212 m631 mu150 sa197 sa231
e1032 e1370 m41 m633 sa186 sa219 tm1236
e1286 e1371 m577 m646 sa187 sa223 ttTi14461
e1365 e1391 m579 m65 sa189 sa229 ttTi36575
e1368 e979 m596 mg43 sa193 sa230 ttTi7618
• 35 alleles
• Different severity for different phenotypes

48. daf-2 receptor: a historical perspective
•1981
•Missouri
•Mutagenesis screen
•Dauer formation

49. What is a dauer?
• Alternative developmental stage
• Thin elongated morphology
• Lethargic
• Super resistant
dauer

50. Favorable
Unfavorable
HIGH Food
LOW Pheromone
Temperature
How do we get dauers?
Dauer
Grow
Don’t Grow LOW Food
HIGH Pheromone
HIGH Temperature

51. Favorable
Unfavorable
Dauer Mutant Screen
Dauer
Grow
Don’t Grow
DAuer Formation Mutants
“daf”
Dauer Constitutive
Dauer Defective

52. daf-1daf-2
daf-10
daf-12
daf-14daf-15
daf-16
daf-18
daf-19
daf-23
daf-28
daf-36 (makes daf-12 ligand)
daf-3
daf-4
daf-5
daf-6
daf-7
daf-8
daf-9 (makes daf-12 ligand)
Epistatic Ordering of daf genes
Insulin Pathway TGF-B Pathway
INCREASED LIFESPAN
Dauer Hormone Synthesis
Sensory Mutants

53. Tools for deciphering daf-2
DAF-2 RECEPTOR
• 35 alleles
• Different severity for different phenotypes
• Need second longevity variable
TEMPERATURE