FCUL - MSc course 13 Nov 2017 · Neuroendocrinecontrol of development Alisson Gontijo CEDOC –NOVA...

Neuroendocrine controlofdevelopment

AlissonGontijoCEDOC– NOVAMedicalSchool Lisbon,Portugal

[email protected]@alisson_gontijo

Drosophilasuzukii lifecyclebyNicolasGompel.

Modified from Lange etal.RoskildeUniv.Based on (Edgar,2006;Layalle etal.,2008;Nijhout,2003;Riddiford,1993;Schubiger etal.,1998).

TIME

Drosophilapost-embryonic development

growth metamorphosis adult


Drosophilapost-embryonic development

TIME

growth metamorphosis pharateadult

adult

AtlasofDrosophilaMorphology:Wild-typeandClassicalMutantsBySylwester Chyb,NicolasGompel

…....

Chen,F.(2016).

PTTH

Ecdysone

Ringgland


Hormonescontroldevelopmental transitions

TIME

Brain

growth metamorphosis pharateadult

adult

Imaginaldiscsarethe precursos ofseveral adult structures

Imaginaldisccells arekept atadiploid stateGrow mitoticallyHave the capacity to regenerate during larvalstage

Imaginaldiscs

RegenerationSlow growth

Tumoralgrowth

Imaginaltissue damage prolongs the growth period ofDrosophilalarvae

From Lange etal.RoskildeUniv.Based on (Edgar,2006;Layalle etal.,2008;Nijhout,2003;Riddiford,1993;Schubiger etal.,1998).

Simpson et al, 1980

Imaginaldiscsproducean inhibitoryimaginalsignal

Halme etal.CurrentBiology20,458–463,March9,2010.

(PTTH)

labial disc

eye antennal discs

leg discs

wing discs

haltere discsgenital disc

wt tumor

pupalarva

Affymetrix

Upregulated?

SignalP?

Candidate

inhibitory imaginal signal

?

labial disc

eye antennal discs

leg discs

wing discs


wt tumor

pupalarva

Affymetrix

Upregulated?

SignalP?

Candidate


?

Ferres%Marco*et*al.,"Nature"2006*

We used an eye tumormodel that delaysthe onset ofmetamorphosis to search for

this inhibitory imaginalsignal

Key:we hypothesized that this signal was ahormoneofpeptidic nature

labial disc

eye antennal discs

leg discs

wing discs


wt tumor

pupalarva

Affymetrix

Upregulated?

SignalP?

Candidate


?

#1

labial disc

eye antennal discs

leg discs

wing discs


wt tumor

pupalarva

Affymetrix

Upregulated?

SignalP?

Candidate


?

labial disc

eye antennal discs

leg discs

wing discs


wt tumor

pupalarva

Affymetrix

Upregulated?

SignalP?

Candidate


?

Klebes etal.Development,2005.

SignalP B-Chain C-Peptide A-ChainC C C CC C

DILP8

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

CG14059encodes an insulin-like peptide we named DILP8

N-DHSSRSYNNIPYC13C14LNQC18EEEFFC24-C

N-SFC3SLERMKKFAMEAC16EHLFQADEGA-C

Chain A

Chain B

DILP8G-protein-coupledreceptor

receptortyrosinekinase

IRIGF1RLGRs

DILP8is the only insulin-like peptide upregulatedintumors

The dilp8responseto eye tumors is conserved

Rel

ativ

edi

lp8

mR

NA

leve

ls

Aprotein trap canbeused to follow dilp8activity

dilp8respondstovariousformsofabnormaltissuegrowth

dilp8isrequiredforthepupariation delayassociatedwithvariousimaginal discabnormalgrowths

dilp8(-/-)larvae aremutagen sensitive

DILP8is required for the abnormal-growth dependent delay ofthe onset

ofmetamorphosis

Is it sufficient?

DILP8is sufficient to delay the onset ofmetamorphosis

DILP8is an inhibitory imaginalsignal

MASOPMasterofpupae!

DILP8,the “masterofpupae”,is aninhibitory imaginalsignal

repair/regeneration

DILP8Environment

intrinsic

wt

PUPA ADULTwt

PUPArepair/regeneration

dilp8 (-/-)

Environment

intrinsic

Developmentalerrors

PUPA ADULT

PUPAdilp8 (-/-)

intrinsic ADULT?

dilp8mutants showgreater intraindividual variability

dilp8(-/-)

dilp8mutants showgreater intraindividual variability

dilp8promotes inter- andintra-individualstability bycontrolling maturation time

repair/regeneration

DILP8

Environment

intrinsic

wt

PUPA ADULT

plasticity stability

??

How does DILP8signal adelay?

repair/regeneration

DILP8

Environment

intrinsic

wt

PUPA ADULT

plasticity stability

DILP8controls developmental timingby inhibiting ecdysone biosynthesis

Inconditions ofdamage/regeneration,DILP8acts upstream ofPTTHtranscription

DILP8inhibitstheecdysone biosynthesispathwayintheabsenceoftissuedamage

Ecdysone defficiency intub>dilp8animals?

Feeding 20HEto larvae supresses thedilp8-dependentpupariationdelay

DILP8increases body weight but notbody size

Stieper etal.,Dev Biol 2008

X

Developmental stability Developmental plasticity

DILP8inhibits imaginaldiscgrowth

FOXO

Thor/4E-BP

IIS

Growth

DILP8inhibits imaginaldiscgrowth by promoting FOXOactivity

Dilp8isakeyfactorpromotingdevelopmentalstability

PTTH neurons

Abnormally-growingimaginal disc

Dilp8

Ring gland

Developmentalprogression

Ecdysone

CNS

Larva Pupa

Howdoesitwork?

Literature:Simpsonetal.,1980;Bryant&Schmidt,1990;Poodry &Woods;1990;Menut etal.,2007;Stieper etal.,2008;Smith-Boltonetal.,2009;Halme etal.,2010;Tennessen &Thummel,2011;Parkeretal.,2011;Garelli etal.,2012;Colombani etal.,2012.

Insulin like-peptide superfamily

Ilps%

Relaxins%Insulin% IGFs%

GPCRs%

InR% IGFR%Receptor%names%

Func6on%

• %Growth%

• %Metabolism%

• %Aging%

• %Reproduc6on%

• %Tissue%remodelling%

• %Neuronal%signalling%

RTKs%

RXFP1/2% RXFP3/4%RXFP1/2% RXFP3/4%

Receptor%type%

Ilp%family%

Andreia Casimiro

SignalP B-Chain C-Peptide A-ChainC C C CC C

DILP8

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

N-DHSSRSYNNIPYC13C14LNQC18EEEFFC24-C

N-SFC3SLERMKKFAMEAC16EHLFQADEGA-C

Chain A

Chain B

DILP8G-protein-coupledreceptor

receptortyrosinekinase

IRIGF1RLGRs

Garelli etal.,Science,2012

Dilp8is asecreted peptide of the insulin/IGF/relaxin-like superfamily

Question:

• DoesDilp8actviaaninvertebraterelaxinreceptor?

VanHiel etal.,Peptides2011

GeneratingDrosophilaRelaxin ReceptorLgr3mutants

5 6 7 8 9 10 11

Lgr3 ChrIII

MB06848

100 bp

ag1 (Δ3.8 kb)

C-terN-ter

LDLα

LRR

7TMTM/SP

G protein coupling

ag1

Question:

• WhatisthephenotypeofLgr3mutants?

0

0.5

1

1.5

2

2.5

3

012345678

p=0.002 p=0.028

Win

g ar

ea F

Aix

104

Win

g le

ngth

FAi

x 10

4

Lgr3 +/+ Lgr3 ag1 Lgr3 +/+ Lgr3 ag1

(19) (52) (25) (66)

Lgr3mutants showincreased intraindividual variability

dilp8(-/-)Garelli etal.,Science,2012

dilp8mutants showincreased intraindividual variability

Conclusions• Lgr3mutationincreasesfluctuatingasymmetry

Question:

• IsLgr3requiredforthecouplingofgrowthtodevelopmentaltiming

PTTH neurons

Abnormally-growing imaginal disc

Dilp8

Ring gland

Developmental progression

Ecdysone

Larva Pupa

CNS

4hregglaying

24hr

Pupariation timing assays

60 80 100 120 140 160 180 200 220 240 260

Lgr3

+/+

Lgr3

ag1

Bx>rpr

Bx>rpr

Lgr3 -/-

Lgr3 +/+DELAY

?

Time AEL (h)

Dilp8

Dilp8

(473)

(367)

(235)

(130)

(298)

(105)

A

A

C

A

A

B

Bx>

rpr

Bx>rpr

Bx>

rpr

Bx>rpr


• Lgr3isrequiredforthecouplingofgrowthtodevelopmentaltiming

Question:

• DoesLgr3actintheDilp8pathway?

PTTH neurons

Abnormally-growing imaginal disc

Dilp8

Ring gland

Developmental progression

Ecdysone

Larva Pupa

CNS

tub>dilp8b/c

tub>dilp8b/c

Lgr3-IR

Lgr3 +/+DELAY

?

Dilp8

Dilp8

Time AEL (h)

dilp8b

dilp8c

60 80 100 120 140 160 180 200 220 240 260

(125)

(92)

(236)

(123)

(55)

(135)

(71)

(39)

B

B

A

C

AB

A

C

AB

Lgr3-IR

tub>

-

tub>

tub>Lgr3-IR

-

tub>

tub>Lgr3-IR

Lgr3 ag1/+

Lgr3 ag1/Lgr3 ag1

Lgr3 ag1/Lgr3 Df(3)arm

>dilp

8a

Time AEL (h)

arm>dilp8a

arm>dilp8a

Lgr3 -/-

Lgr3 -/+DELAY

?

Dilp8

Dilp8

Lgr3 ag1

Lgr3 Df(3)BSC321

Lgr3 +/+Lgr3 control

Lgr3 mutant

Lgr3 null (deficiency)

Chr3R25710k

1 kb

(56)

(112)

(42)

B

A

A

80 100 120 140 160 180 200 220



• Lgr3mutantsareinsensitivetoDilp8activity

Question:

• WhereisLgr3expressed?

sfGFP repair construct

+Lgr3

sfGFP::Lgr3 [ag5] PTC-

sfGFP::Lgr3 [ag6-9] PTC+

sfGFP

Lgr3 CDS (exon2)

Strategy:CRISPR/Cas9-mediatedhomologydependentrepairGFPknock-in


+Lgr3



sfGFP

Lgr3 CDS (exon2)

sfGFP C-terN-ter

LDLa

LRR

7TMTM/SP

G protein coupling

ag6-9

ag1

Strategy:CRISPR/Cas9-mediatedhomologydependentrepairGFPknock-in

sfGFP::Lgr3[ag5] (PTC-)

DAP

I n

c82

sfG

FP::L

gr3

sfG

FP::L

gr3

c sfGFP::Lgr3[ag7] (PTC+) sfGFP::Lgr3+arm>Lgr3-IR

A

R

MIL

PIL


+Lgr3



sfGFP

Lgr3 CDS (exon2)



• Lgr3mutantsareinsensitivetoDilp8activity• Lgr3isexpressedintheCNS

Question:

• Whatdoestheneuroanatomy ofLgr3neuronstellus?

Lgr3neuronsdonotinnervatetheringglandandLgr3isnotexpressedinneuronsknowntocontroldevelopmentaltiming

Siegmund andKorge,JCompNeurol,2001.

DAPI anti-Dilp5 sfGFP::Lgr3

PG

IPCp

CA

CC

PG ao

brain

b

PTTH-HA sfGFP::Lgr3

PIL

MILVNC

f

brain

d

Insulin producing cells + PIL neurons

DAPI anti-Dilp5 sfGFP::Lgr3

D

V

A

P

L

R

IPCs

cbcb



• Lgr3mutantsareinsensitivetoDilp8activity• Lgr3isexpressedintheCNS• Lgr3neuronsrevealanewcircuitcouplinggrowthtodevelopmentaltiming

Question:

• IsLgr3requiredintheseneuronstotransmittheDilp8signalfromtheperipherytotheringgland?

+PB

S

+EM

S

+EMS

Dilp8

?

DELAY Lgr3 +/+

elav>Lgr3-IR

Lgr3 neurons

Lgr3 neurons

Time AEL (h)80 100 120 140 160 180 200 220 240 260

+EMS

Dilp8

(40)

(34)

(23)

(57)

(38)

(32)

B

AB

A

C

AB

AB

-

arm>Lgr3-IR

elav>Lgr3-IR

-

arm>Lgr3-IR

elav>Lgr3-IR

0

1

2

3

4

5

6

elav>

>Lgr3a--

Lgr3 ag1/ag1Lgr3 ag1/+

p=0.005

(14) (21) (24)

Win

g ar

ea F

Aix

104

0

2

4

6

8

10

Lgr3-IR-Lgr3-IR

elav>

p=0.0006

p=0.0005

(24) (24) (20)

Win

g ar

ea F

Aix

104




• Lgr3isrequiredintheCNS

Question:• WhichoftheLgr3neuronsisresponsibleforconveyingtheDilp8delaymessage?

Lgr3 5 6 7 8 9 10 11

Lgr3 ChrIII100 bp

GMR17G11 GMR19B09

4321

http://flweb.janelia.org/

Lgr3 5 6 7 8 9 10 11

Lgr3 ChrIII100 bp

GMR17G11 GMR19B09

4321

b80 100 120 140 160 180 200 220 240

+PB

S

+EM

S

80 100 120 140 160 180 200 220 240

+PB

S

+EM

S

c

GMR19B09>

X

X

Lgr3-IR +EMS

GMR19B09>

Kir2.1 +EMS

(72)

(29)

(77)

(33)

A

A

C

B

GMR19B09>

GMR19B09>Lgr3IR

GMR19B09>

GMR19B09>Lgr3-IR

(75)

(75)

(80)

(70)

A

A

C

B

GMR19B09>

GMR19B09>Kir2.1

GMR19B09>

GMR19B09>Kir2.1

Time AEL (h)

DAPI GMR19B09>myr::tdTomato sfGFP::Lgr3d

PIL

A

R

PIL

PILarb

PILarb

PIL

PILarbfMIL

f brain

VNC

*

GMR17G11-myr::tdTomato sfGFP::Lgr3

PIL

PIL

A

L MIL

f brain

VNC

*PIL

PIL

MIL

f brain

VNC

*PIL

PIL

80 100 120 140 160 180 200 220 240

+PB

S

+EM

S

Time AEL (h)

(40)

(21)

(57)

(29)

A

AB

B

B

-

GMR17G11>Lgr3-IR-V22

-

GMR17G11>Lgr3-IR-V22

GMR19B09-Gal4>myr::tdTomato ∩sfGFP::Lgr3 ~ 70 neurons

DAPI MZ699>myr::tdTomato sfGFP::Lgr3

PIL

A

R

#5PIL

#5

e

PIL

#5

f

Time AEL (h)80 100 120 140 160 180 200 220 240

+PB

S

+EM

S

(73)

(29)

(77)

(33)

A

A

C

B

MZ699>

MZ699>Lgr3-IR

MZ699>

MZ699>Lgr3-IR

MZ699> X Lgr3-IR +EMS

Itoetal.,CellTissueRes.1997Oct;290(1):1-10.

PILneuronsareasubsetofMZ699#5neurons

MZ699-Gal4>mCD8::RFP � GMR19B09-LexA>mCD8::GFP

a

f

PI

brain

seg

VNC

brainf

PI

brain

seg

VNC

brain

a

f

PI

brain

seg

VNC

brain

Lgr3isrequiredinPILneuronsor7otherbilateralVNCneurons

MZ699 � GMR19B09

Conclusions• Lgr3mutationincreasesfluctuatingasymmetry• Lgr3isrequiredforthecouplingofgrowthtodevelopmentaltiming


• Lgr3isrequiredintheCNS



• Lgr3isrequiredintheCNS• Lgr3isrequiredinagroupof9bilateralneurons

Question:• DoesDilp8induceanycAMP-activityinvivo?

Lgr3inducescAMP responseinHEK293cells

VanHiel etal.,2014. Ijima-Ando&Yin,2005

MIL

f

brain

clock neurons

brain

vnc

clock neurons

fclock neurons

clock neurons

brainbrain

* *

*

*

A

L

A

L

MIL

f

brain

clock neurons

brain

vnc

clock neurons

fclock neurons

clock neurons

brainbrain

* *

*

*

MIL

f

brain

clock neurons

brain

vnc

clock neurons

fclock neurons

clock neurons

brainbrain

* *

*

*

CRE-Luciferease sfGFP::Lgr3

w1118

tub>dilp8b

seg seg seg

seg seg seg

Dilp8activatescAMP-signalingreporterinPILneurons

f

PIL neurons

A

L

f

f f

tub>dilp8b

MIL neurons and other Lgr3+ cells

A

L

f

MIL

clock neurons

f

MIL

clock neurons

f

MIL

clock neurons

f

MIL

clock neurons

DAPI CRE-Luciferase sfGFP::Lgr3

a b

Dilp8activatescAMP-signalingreporterinPILneurons



• Lgr3isrequiredintheCNS• Lgr3isrequiredinagroupof9bilateralneurons



• Lgr3isrequiredintheCNS• Lgr3isrequiredinagroupof9bilateralneurons,twoofwhicharetheDilp8-responsivePILneurons

Dilp8isakeyfactorpromotingdevelopmentalstability


Dilp8

Ring gland


Ecdysone

CNS

Larva Pupa

Howdoesitwork?

Literature:Simpsonetal.,1980;Bryant&Schmidt,1990;Poodry &Woods;1990;Menut etal.,2007;Stieper etal.,2008;Smith-Boltonetal.,2009;Halme etal.,2010;Tennessen &Thummel,2011;Parkeretal.,2011;Garelli etal.,2012;Colombani etal.,2012.

PTTH neurons


Ring gland


Ecdysone

Larva Pupa

CNS

Dilp8

PTTH neurons

Literature:Simpsonetal.,1980;Bryant&Schmidt,1990;Poodry &Woods;1990;Menut etal.,2007;Stieper etal.,2008;Smith-Boltonetal.,2009;Halme etal.,2010;Tennessen &Thummel,2011;Parkeretal.,2011;Garelli etal.,2012;Colombani etal.,2012;Garelli etal.,2015.

Lgr3 neurons

Dilp8-Lgr3pathwaypromotesdevelopmentalstability

PTTH neurons


Dilp8

Ring gland


Ecdysone

Larva Pupa

Relay 1?CNS


Lgr3 neurons


PTTH neurons


Dilp8

Ring gland


Ecdysone

Larva Pupa

Lgr3 neurons Relay 1?

Relay 2?

CNS



Question:• IfLgr3isnotthereceptorforDilp8whatisitthen?

Question:• WhichcellsurfacereceptorsbinddirectlytoDilp8?

c

a

0

1

2

3

4

5

-10 -5 0 5 10

Log2 (INS / Glycine)

-Log

10 (F

DR

-adj

uste

d p

valu

e)

SIL1

INR

0

1

2

3

4

5

-10 -5 0 5 10

Log2 (Dilp8 / Glycine)

-Log

10 (F

DR

-adj

uste

d p

valu

e) DRL

NRG

SIL1

INRCTLH2

ITA3

b

Dilp8

+

TRICEPS

+

=

digestion purification mass

spectrometry

Drosophila SL2/DL2

cells

N-glycosylated receptor binding on

Cell surface

=

statistics

x Dilp8

x Glycine (- control)

INS (+ control)

= n?

= n?

= n?

Ligand-ReceptorCapture(LRCCaptirec)assay

c

a

0

1

2

3

4

5

-10 -5 0 5 10


-Log

10 (F

DR

-adj

uste

d p

valu

e)

SIL1

INR

0

1

2

3

4

5

-10 -5 0 5 10


-Log

10 (F

DR

-adj

uste

d p

valu

e) DRL

NRG

SIL1

INRCTLH2

ITA3

b

Dilp8

+

TRICEPS

+

=


spectrometry

Drosophila SL2/DL2

cells


Cell surface

=

statistics

x Dilp8


INS (+ control)

= n?

= n?

= n?

c

a

0

1

2

3

4

5

-10 -5 0 5 10


-Log

10 (F

DR

-adj

uste

d p

valu

e)

SIL1

INR

0

1

2

3

4

5

-10 -5 0 5 10


-Log

10 (F

DR

-adj

uste

d p

valu

e) DRL

NRG

SIL1

INRCTLH2

ITA3

b

Dilp8

+

TRICEPS

+

=


spectrometry

Drosophila SL2/DL2

cells


Cell surface

=

statistics

x Dilp8


INS (+ control)

= n?

= n?

= n?

rela

tive

mR

NA

expr

essi

on

leve

l (rp

49 =

100

)

0

0.05

0.1

0.15

0.2

InR Drl Lgr3


PTTH neurons


Dilp8

Ring gland


Ecdysone

Larva Pupa


Relay 2?

CNS


PTTH neurons


Dilp8

Ring gland


Ecdysone

Larva Pupa


Relay 2?

CNS


InRDrlNrg


IntegrativeBiomedicineLab

Intheflywetrust

Collaborators:

TakashiKoyamaIGC,Portugal

EstherCaparrosUMH,Spain

Yanel VolontéCONICET,Argentina

ThomasUhlmannDualsystems Biotech,Switzerland

AlissonGontijo

AndreiaCasimiro

MarciaGarcez

MariaJoãoCruz

CatarinaNunes

FabianaHeredia

AndresGarelli(CONICET)

AndreMacedo

AngelaDias

Siegmund andKorge,JCompNeurol,2001.

pars-intercerebralis-ringglandaxis Hypothalamic-pituitaryaxis

IntegrativeBiomedicineLab

IntheflywetrustEmail:[email protected] Twitter:@alisson_gontijo

iNOVA4Health

Fabiana HerediaAndresGarelliAndreMacedoAngelaDiasEdnilson VarelaBenilde PondecaJoanaPereirinhaRaquelMonfardiniFilipeViegasAnaLealClaudiaBelémGiseleA.Cardoso

PreviousmembersYanel VolontéAndreia Casimiro

INIBIBBFacundo P.Spalm

AcknowledgementsRibeirolab,CCUVasconcelos lab,CCUMirthlab,IGC/MonashUCordeiro lab,UofGlasgow,UKBloomington,VDRC,JFRCCCU&CEDOCflyfacilities

FCUL - MSc course 13 Nov 2017 · Neuroendocrinecontrol of development Alisson Gontijo CEDOC –NOVA...

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