infusion of egf blocks locomotor activity in hamsters · sleep in worms! egf and egf-r! target...

Infusion of EGF blocks locomotor activity in hamsters

Running wheel assay

Period of infusion; therefore it’s reversible

TGFα, an EGF like factor, disrupts sleep-wake behavior

Mouse EGF Receptor mutant is defective in light response

Together, these experiments suggest EGF is necessary and sufficient for activity/sleep cycle

Sleep in worms

EGF and EGF-R

Target neuron - ALA

Neuropeptides and receptors

CHROMOSOME_IIcctaagcctaagcctaaaatagtgactctggcagttctctaaaataagtgactctggcagttcaccaaaaattgtgactctgaccgttcaccaaaaatagtgactctgaccgttcaccaaaaatagtgactctgaccgttcacaaaaaatagtgactctgaccgttcaccaaatatagtgactctgaccgttcaccaaaaattgtgacaatgaccgttcaccaaaaattgtgactctgaccgtcactatttttattgaactgccagagtcactatttttagtgaacttccagagtcacaatttttagtgaactgccagagtcactatttttagtgaactgccagagtcacttattttggtgcactggggtgggtcacgcccccagttctcagttatgggtactctgatccactcgggacccactttatcgtgttccccgtgcctcatttaccctagagcttcctcctttacctctcctctcgctatctctaacattccaatggaaactcctatttgaattaccgccaccgatgtgcccgacgcgacttactgttagcccttgttttgcacaaatctgttggcttccatatttaaaagttaattaatgacccaatgttctttttttctctaaatctccacaagatgttctgttttccctactggacactatcgttcactgcgtctcaccaattcacattgtctctactttaccttttttgtcatagtacacgttcgccaacggtgtcgacggccaaatgctttgggcagcgtttgctttttttataattagttttattttattaaaacaatagctctaaagtttacaagtcatttgttataggctaaatgagttatgtctaataagtaatttgaactagatacttccgtgtaagtgacaatgtatcggaaaagtcctcaaagtgcgatgtagaagttcacatgtactttgtttggcatgttagtaaaagagccagtatgctgattcattttatattctatatactcatgtaatatgcccatgtaaggtttaattccaaaaatatgagcgtgttctattttataatattttactaaaatacctttcagttaattgcactcaaatttgttgttcttcattctctcgttatgatttaatcttattgcgtcaaggtcattattttaggtccattagttatcgatctgaaacatgttgttgtatttttctattcttgtgagctcaggacacctcatacaactccagagaaaatgtgtctcattattcttgtcttttttcaagatctaatcaattttctacattaacgacgtttttgtcgttctgcttctttttttcgttcgtttgtctcgtccatcagctgtccactcatttctctcccactcactaggcagtgctttgtttggttccgattggcagctggctgcagggcctgcatctcttctatgtctctcatttacttgcattcttttcttcgttaatttttgttatgatatttaaacgggaagaagagtttgtggttcttctttttataatcactaaaacttttggataagtaacaattttctgataaaatattttcacggcgaagaaaaaagaaaaagaagagtagtttttgcacgttttcatataattattttcgttgatcaaatgttcttctggagttttctaataaatttcttatcgactttttttcagaaatttttctcaacttgtcatgtcaatggtaagaaatgtatcaaatcagagcgaaaaattggaagtaagttctttataatttcatttatatactataagttttctcgatcacaggagaaacaaaaacaacagacaacacaaaaaacaataaaacaatattgctctagtaatcaatagtgttgtaaagagggaagaaaattgttatctgtgtagcagtcaacgttgattgagatgttgtgtttgactatagagttgaaaataataacttcaaacttgcaagtcatgacttatcaaacactgccggaacttattctggatcaaaggaaagttgtccaactgtagagtcatgtttttcaaaagaaaacacaatttttaagtataaatattttgaaaaagtatgttttagaagtatgtcaaattaaaaaaaaaatccttggttaaaaaatgatttttttggatatatgtgtatttttaactaaaaatatatactttacatatatattttggcgcagttatttgatctataaatcaaactttttgatagacatttttttatatttacaacaactagggttgttatgaaaacgcctattattctacaaactaaattattttaatcatacattccccactatctaaaaactaatgcaattttcagattttgtcatgtaaatgggtaggatgtctcaaatcaacagaagtgttcaaaacggttgaaaagttattagatcatgttacggctgatcatattccagaagttattgtaaacgatgacgggtcggaggaagtcgtttgtcagtgggattgctgcgaaatgggtgccagtcgtggaaatcttcaaaaaaaggtatttttaatttaatgtgcattttataatataaattcttcagaaagagtggatggagaatcacttcaaaacacgtcatgttcgcaaagcaaaaatattcaaatgcttaattgaggattgccctgtggtaaagtcaagtagtcaggaaattgaaacccatctcagaataagtcatccaataaatccgaaaaaaggtattcacaatttgcatgatattgttataatctaattttcagagagactgaaagagtttaaaagttctaccgaccacatcgaacctactcaagctaatagagtatggacaattgtgaacggagaggttcaatggaagactccaccgcggtaagtgtgtttctttaaaaattacttccttttttcaattgtttgaaattaacaagaaacctgttggagcgtatttctgaacttttaaatcgaaaatatcatttgcaaaaaaacttgaaaattgagaaacttttttaaaagtggagtagcgtctgcgggtttttttgccctaaatgacagaatacatacccaatataccgaatataaccgtaataaaattatgcgatttttatttttatttttcatgaatgttaggggcaaaaaacccacatgcgctactccgccttaagaagaatcagctgtgagcactatccactatacattggaaatttacaaataaaatagagattaagtaatataatttttaagggttaaaaaaaagactgtgatatactatgatgatgggccgaggtatgtatttccaacgggatgtgcgagatgcaactatgatagtgacgaatcagaactggaatcagatgagttttggtcagccacagagatgtcagataatgaagagtacgttgttttgcaaattgattaaaagtggagtagcgtcagttaaaaactctaacatgtcttaggtttttcaaaagtttggtcaaagttttggcaaactgccaacttcttgaaaacttcgttaaaaaaattcttgaaatgatttgaaaatttgtattatgttattctcttatttctgcactattctatatggcgctactatacttttaattgatttcttgaaagcagttcaataataattaattttagagtatatgtgaacttccgtggaatgaactgtatctcaacaggaaagtcggccagtatggtcccgagcaaacgaagaaattggccaaaaagagtgaagaaaaggctatcgacacaaagaaacaatcagaaaactattcgaccaccagagctgaataaaaataatatagagataaaagatatgaagtaagtcgaaattgacaaacagtggtttttgtttaagtttattgcgaaatattcaaaattagacatgttaaaattttgcgagataatctaaagattaggtatacagattttttcatgtaaagttacattcatcaaaatttttgtgttcaccaaattagacaaaaaatgttagttacacagtatatttattttttatatcaataaaacctttttcagctcaaataaccttgaagaacgcaacagagaagaatgcattcagcctgtttctgttgaaaagaacatcctgcattttgaaaaattcaaatcaaatcaaatttgcattgttcgggaaaacaataaatttagagaaggaacgagaagacgcagaaagaattctggtgaatcggaagacttgaaaattcatgaaaactttactgaaaaacgaagacccattcgatcatgcaaacaaaatataagtttctatgaaatggacggggatatagaagaatttgaagtgtttttcgatactcccacaaaaagcaaaaaagtacttctggatatctacagtgcgaagaaaatgccaaaaattgaggttgaagattcattagttaataagtttcattcaaaacgtccatcaagagcatgtcgagttcttggaagtatggaagaagtaccatttgatgtggaaataggatattgattttataacgtgtaattgagttttggccaaaaaggtatggaaaggtggctgtttagttatatatttttctattatttatttgaaacatgcaaaattgaagtgaacaataagtgatgttcatggaaatttaaactgttttatgatacttttttgagaaattgaaaaatctgttcattttagaaacaatgtccacatggttctaagagctaaaatttttattttcatccatttagagtactttctcttttagagtacggccccagagcgatgttagaaacctgagatcggtcaacacagaccgttaattttgggaagttgagaaattcgctagtttctgacacgaatttcagctaataccaaataatgtgcaattgcattttgcatgtcagcattcagcattcatacaaaaatttcaaagagccaacttttcatacgtttatggtcaacactgtatgtgttacattgaacttttttaaattgtattatttcatatttgaatcatttccatgccattttcaaatctttttttaacaaaaatttagtttcaaagttttaaatttaggtgaaaacttgctacaaaataacacattctttagtcgtttcaattgctatctatccgcagactgcaattttgttttcccacaaccattcacacaataaataagggtataaagttttgttcatataacacatttcaatactaacatttcaattttgaacaatttttctaaacttatttcccttcgcccaaacgtcattcaacattctttgtacaaaacattaccattatagaaaatctcatttttccactatttcatttatttttattgttccgcgataaatataaataaacatttacgtgttccgagttcaaagttttaccacgtttcagaataaaggaatcggaggggggggggggtgaaaaaatcatttcaacaaatcagaatttctcaaaatgtgagttttatcattttcattgttagaatcacgattagcttatattgaacaataacaataatttaatcccattcattccatatcttctcatgatgaaaaaataagcattattcgttttcttttgacgcgtttgatagggttctgccagcgccgaccaatattcttctcaattagattttccagaactgcactaactgaaaattgttttagtaaatagaacaaaactgactattagttcaatattataattttacttccctattttctgacagcattttgcaaatactctttacagtcattcttgtattttgacaacaaaattcgaatttaaaatttcatttttcttttaaaatatcagtacactcctggtagaacaaatattttatttcaatgtttatgtatgagtagaaaagttaaacagaactattatttggcatcaatttctgttttttttttcatctaaagtatggcatttattagcaaaaaatacatgcttgaacaggaataactattaatttcctatagcgacccaaaaacaaccaaaaattgttttaaataatttttttttggtcgacttccatagttatgagtggcaaaaactgagtaattgtcaccttttgacagtaaataaagaaattttcaaaaaatttttgaaaagttttattatgctattcgatcattttggcaccatgtaggctttaacaccccactggcgctactccgcatttaaaagtgatttataaaaagtgtaaggaatactttgctccaattttcgtctagtgcatggcgtacccatattttgttcatgttgtgtctcttagcctctcctcggaggcgagtttccatagctacccacaaagaatttatttttctatgtatgcttcgttggctccaccttcattttctcagtcgtttctcagttttctcatgaattctctttttgttcgtttttagaatagctatcgttttaccacgaatgcgctttttcagttattaagaaaacagttttttgtagtacatttttcatagtctgaactttcagcatggaaatagtaaaaacaatcattccacacaaccgttcatacattgaaccgcccataccatctgccaccgagtggcttgcaagtaagttgttttgtctaaagacataatttctgaaagaaatctatccgatgagcggaaaataaacagaagttgtttttaagaagactttgatgcaattacaaaaatatatggctatgtgcgtaaaaattagacgtgactcgtcataaagaccatataggtctacaaaggcgtttcgagtcttataaaagttcgttcaaactttttcggggtcttgttaaaagcacaccaacaatacgttaaaggatttataaaattgcaagtattcgtctcaattgcaatactttggaagccgaattttggcaagatattggtaaaacaggtacattacaatagctatttttggtaaaatgtactaggtatcttgtaatgagttgcgcttgcctcataggcatacatctaccgtatattctttactagtgctgcaggcagcactaatttctaggcccttttttaatgcagtactattagagactgcagtactactggagatgcagcgctaatagagaatatacggtatgtagatgcctagaacgctaaggttgttttattcaaataaatttcttagaaaggctaatattatttttaaaatcaagatttaagttaattaacacgacctcgtattctatatttcagatatgtcagttatgcatgtgtcatgtttaaccattgcttgtgtatttgttgctatcacatttttgtcatcatttttccatttattttttgtattgaaatatgtgtctaatgagaggatacgcaatgatatgtacgcattgatatttatgtttcccgtgagtttataaatggaattatgaagagtttggaaaatatattttagataaccacatttgcaagtttagtaggaatgttcataccaagagcggctattttcctttatgcagtttcccttgtgtaagtgtaccttaacaccacaaacagaaaaatgaactttattgatttgttccagatatttcatgtttaccctcttcataatggtcactcttttattcaacatttttggaggtcggcaagaaatgagtgcctacttgctccaaagaaatattcgagttaatttcactgttccaccactctgttttttcaagttcctgccaactgtagaaagtacagagtgagctcgaaaatcttatttttgtctttctaatatattttccagtcaaaacctacgtcgaatcgagtggttggtttttcaaactcctattatacgaactcttttagagttagtctcagtcgttgtgtctatggaacaagaaggaagacgagaaagcgtgtaagaccatgaaatgttttttgtgtgcgcaacgtagtgcgaaaagttagttatttggagaggaagttatttgaaaagaatctttagaagatattgatattttatattctgtatgtaggcgctgtggcaaattctcgacttcacgggagaagcgcaaattttgtgctggtttccataatgcgctcgaaaaatgttgtatatttacattattcaaatctcaccgattaaagttgaatttcagttggttcgtattctctcaactcatggcactactctcgatgtgcattgccttttatggttgctacgttatggttcctcttggaagagaaaagcatgctccataccgattcgattttctatttcgtacatgtgacattgctcagtgtatttacacaattcaaaagtttgtctttgagtttgctgctgcagttggcttgatcacttcggatcgataccttccagctgctgcgaaggcattgtgtaagttcagaagtttaaatggagataggaatgtttcgattttcgtttataaaaacacctgttaatattaaattgttgacgtcatagctgaccattcatgcttctttgtctgattgtaattatataattgtaaaaatacaaacaagttaaccgaaaaagttctctgttacgtttgcggtaaattagaactttatgttgaaagatatcagcttaaagttaaaattttttgaaaattaaattttggctagaaattcaatttttctgataaaaagaaagtttggtgggaaatttaatttcattgatagaaaaactgaagtttgtgttttctgagaattaaaaaacagcgggcaatgaagattatgtctttgtaaatttcaatttaaaaatattccagggtgggcatcgtttatgtgcacctgggagatgatgctcctctctgctctttgctcatattgtttgcgtccagccaagtgtaaattttttgatctttaccctggaaacgatatgccagctttatcggcaagagatggttcaaactcccgtgttccgtcattttctcgacgtctatctattgaatatgagccacgaattgctggagtgatgttggagccgccgtcaagaagttcactgagcattactcctagggataaaatagaagacccaacaaccgtatcgtattttgctgataattttgattctttatcgcaaatccaaggacaataattgaacttcctgccattatttcattgctcaatttacactattcttaactttttgacatgaattaaatctttttaaactgagtctaagcgtattattgtatcgtattttccccttctgatgtattcatttacttgtatttttgaaacccatcccagtgatacaactacccaattttcctgtgccatgtttcttgaaacaaatcaaatgtgat!

6  

C.  elegans  SLEEP:  molecules  &  circuits      

graphic  by  Chris  Grove  [WormBase-­‐Caltech]  

Elly  Chow  Julie  Cho  Cheryl  Van  Buskirk  Erich  Schwarz  Daniel  A.  Lee,  David  Prober  

302 neurons

Paul Sternberg, Cal tech

Neuropeptide signaling network promotes EGF-dependent sleep

Sleep dampens sensory response by

multilevel modulation of a sensory-motor circuit

1

2

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Vulval development

PLCγ [PIP2]

Ovulation

ITR-1  IP3 Receptor

SLI-1  

Cbl LFE-2  IP3K

LET-23  EGF-R

LIN-3 EGF

[DAG]

Y~P   Y~P  

Wild-type adult worms are almost constantly moving

Cheryl Van Buskirk Van Buskirk & Sternberg Nat Neuro 2007

EGF expression severely inhibits locomotion

(no movement unless perturbed)!Cheryl Van Buskirk

WT  lethargus            hs:EGF  adult  

unperturbed  perturbed  

Octanol  latency  

hs:EGF induces sleep-like behavior outside of lethargus

cessaQon  of  feeding    and  locomoQon  

 reduced  sensory    responsiveness  

 quick  reversibility  

hatch!

embryogenesis L1 ! L2 ! L3 ! L4 ! Adult!

molt    molt            molt                molt  

Cheryl Van Buskirk

second

s  unQ

l  respo

nse  

EGF promotes quiescence during molting via ALA neuron

Time  (min)  

Velocity  (m

m/sec)  

0                  50              100      150              200                    0              50                100                150                200  

0.20  

0.15  

0.10  

0.05  

0  

-­‐.05  

0.20  

0.15  

0.10  

0.05  

0  

-­‐.05  

N2   let-­‐23(sy12)  

plc-­‐3(tm1340)   ALA-­‐  

Wild-­‐type,  intact  

Wild-­‐type  ALA-­‐ablated  

EGF-­‐receptor  mutant  

PLC-­‐γ  mutant  

Van Buskirk & Sternberg Nat Neuro 2007

EGF promotes quiescence during molting via ALA neuron

Time  (min)  

Velocity  (m

m/sec)  

0                  50              100      150              200                    0              50                100                150                200  

0.20  

0.15  

0.10  

0.05  

0  

-­‐.05  

0.20  

0.15  

0.10  

0.05  

0  

-­‐.05  

N2   let-­‐23(sy12)  

plc-­‐3(tm1340)   ALA-­‐  

Wild-­‐type,  intact  

Wild-­‐type  ALA-­‐ablated  

EGF-­‐receptor  mutant  

PLC-­‐γ  mutant  

Van Buskirk & Sternberg Nat Neuro 2007

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Vulval development

PLCγ [PIP2]

Ovulation

ITR-1  IP3 Receptor

SLI-1  

Cbl LFE-2  IP3K

LET-23  EGF-R

LIN-3 EGF

[DAG]

Sleep

Synaptic Release UNC-13, etc.

EGF signaling mediates stress-induced sleep

Time  (min)  

Velocity  (m

m/sec)  

0                    50              100                150                200                0          50                100                150                200  

0.20  0.15  

0.10  

0.05  

0  

-­‐.05  

WT   let-­‐23(rf)  

lethargus  behavior  EGF/EGFR  

STRESS  

stress-­‐induced  sleep  Van  Buskirk  Lab,  Cal  State  Northridge  

The single ALA neuron is necessary for  

EGF-dependent sleep

~ALA-­‐ablated  animals  are  insomniac  ~Expression  of  EGFR  only  in  ALA  is  is  sufficient  for  EGF-­‐induced  sleep  ~ALA  synapses  not  necessary  for  funcQon  

   Cheryl Van Buskirk

Micro-dissection & Transcriptional profiling of single ALA neurons

L4  

L4  Lethargus  4-­‐5hr  

Single  cell  RT-­‐PCR  cDNA  amplificaQon  9  cells  (2  pools)    

Igor  Antoshechkin,  Caltech  Jacobs  Genome  Center  

Elly  Chow  Erich  Schwarz,  Miriam  Goodman,  Mihoko  Kato  Schwarz,  Kato  &  Sternberg  [PNAS  2012]  

Erich  Schwarz  

ALA enriched genes: 10 neuropeptide genes

FRMFamide-Like Peptide

Ex

pre

ssio

n L

eve

l

Enrichment

8110  protein  coding  genes  detected  in  ALA    

ALA-­‐enriched  neuropepQdes  (>12-­‐fold  over  whole  larvae)  

Validation of expression in ALA neuron using enhancers predicted from “ALA motif”

FLP-24

FLP-7

FLP-13

VPSAGDMMVRF!!

SPMQRSSMVRF!TPMQRSSMVRF!SPMERSAMVRF!SPMDRSKMVRF!

!

AMDSPLIRF!AADGAPLIRF!APEASPFIRF!ASPSAPLIRF!SPSAVPLIRF!ASSAPLIRF!SAAAPLIRF!

VPSAGDMMVRFG!

VPSAGDMMVRFamide!

KRVPSAGDMMVRFGKR!

FLP-13

NLP-8

FLP-24

FLP-7

NPR-7

NPR-22

sleep EGF

ALA neuron

neuropeptides receptors

FRPR-3

?

STRESS

EGFR

Elly  Chow  

sleep neuropeptides receptors

sleep neuropeptides receptors

Elly  Chow  

35°C, 30 min recover RT, 10 min

score

4 neuropeptides necessary for EGF response

Qui

esce

nce

Time (min)

WT

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

Elly  Chow  

Qui

esce

nce

Time (min)

Multiple receptors are necessary

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

Elly  Chow  

FLP peptides increase response latency

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

sleep neuropeptides receptors

Elly  Chow  

receptor mutant block peptide overexpression

FLP-13

NLP-8

FLP-24

FLP-7

NPR-7

NPR-22

sleep EGF

ALA neuron

neuropeptides receptors

FRPR-3

?

STRESS

EGFR

Elly  Chow