Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis

Christian Alexandro Clement

Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis

Christian Alexandro Clement

Cellebiologi og Fysiologi

Abstract

Denne PhD afhandling har fokus på cilier og deres sensoriske funktion hos pattedyr. Mere specifikt spiller

Hedgehog (Hh) signaleringsvejen, der virker via det primære cilium, en afgørende rolle for udvikling,

differentiation, cancer og muligvis også stamcelle vedligeholdelse. Defekt ciliedannelse eller ciliefunktion er tæt

knyttet til udviklingsdefekter og sygdomme der betegnes "ciliopatier".

Hovedopgaven i denne afhandling var at undersøge rollen af det primære cilium i koordineringen af Hh signaling

under stamcelledifferentiering og hjerteudviklingen hos mus. Vi viser her, at humane embryonale stamceller

(hESC) samt museembryonale carcinoma-stamceller (P19.CL6) danner primære cilier, hvortil essentielle Hh

proteiner, Gli2, Ptc1 og Smo lokaliserer. Inhibering af Hh signalvejen med KAAD-cyclopamine i P19.CL6 celler,

hæmmer dannelsen af synkront bankende minihjerter. Knockdown af det primære cilium i P19.CL6 celler ved

nukleofektion med plasmider, der udtrykker Ift20 og Ift88 siRNA, reducerer signifikant dannelsen af bankende

minihjerter og efterligner effekten af cyclopaminbehandlingen. In vivo forsøg viste at E11.5 Ift88-/- mutant mus

(der ikke har primære cilier) har alvorlige endokardiale pudedefekter, underudviklet trabekulering, udvidet

perikardial hulrum samt forkortet og misdannet hjerteudløbstrakt. Disse resultater foreslår en mulig rolle for

det primære cilie i koordineringen af Hh-signaleringsvejen under stamcelledifferentiering og hjerteudviklingen.

Ydermere støtter præliminære forsøg med chimeramus hypotesen, at det primære cilium er vigtigt for

hjerteudviklingen. Dette er bedømt ud fra distribueringen af enzymatisk mærkede vildtype og Ift88-/- ES celler,

hvor vildtypecellerne lokaliserer til hjertekamrene, som stort set er fri for mutant ES celler. Dette betyder at det

primære cilium er nødvendig for dannelsen af hjertekamrene.

Den sekundære opgave i denne afhandling var at undersøge rollen af progestosteronsignalering i de kvindelige

reproduktionssorganer samt rollen af det primære cilium i udviklingen af human pankreas og cancer.

Tilstedeværelsen af progestosteronreceptoren i den nedre halvdel af de bevægelige cilier i æggelederne

indikerer en mulig sensorisk rolle for de bevægelige cilier i progestosteronsignaleringen som koordinator for

postovulatoriske begivenheder. I undersøgelserne med den humane pankreas, fandt vi, at den udviklende

eksokrine dukt danner primære cilier på op til 20µm, der projicerer ud i duktlumen fra duktepithelet både under

embryonal (uge 7.5) og føtal udvikling (uger 14 og 18). Analyserne med vævssnittene viste endvidere, at

niveauet af Hh-komponenterne, Gli2 og Smo, kraftigt stiger under den føtale udvikling og er fraværende under

den embryonale udvikling. Denne forøgelse i ciliær Hh signalering foreslår en rolle for det primære cilie i

koordineringen af Hh signalering i modning af det eksokrine duktsystem. Ydermere ses der i kulturer af humane

pankreasdukt adenocarcinoma cellelinier, PANC-1 og CFPAC-1, at både Ptc, Smo og Gli2 lokaliserer kraftigt til de

primære cilier og at Hh signalering er kraftigt opreguleret i cancercellerne i fravær af Hh-stimulering. Disse

resultater tyder på, at ciliær Hh-signalering kan spille en rolle i tumordannelse.

Afhandlingens resultater støtter konklusionen, at både motile og primære cilier spiller en afgørende rolle som et

sensorisk organel under udvikling og i vævsfunktion.

Originalsprog	Engelsk

Udgivelsessted	Biologisk Institut
Forlag	Museum Tusculanum
Antal sider	191
Status	Udgivet - 15 nov. 2009

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http://www.bi.ku.dk/bibliotek/phd/Christian%20Alexandro%20Clement.pdf

Citationsformater

@phdthesis{608194306d6a11df928f000ea68e967b,

title = "Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis",

abstract = "This thesis focuses on cilia and their sensory function in the mammalian organism. In particular, the Hedgehog(Hh) signaling pathway functions via the primary cilium and plays a unique role in development, differentiation,cancer and possibly in stem cell fate. Defects in primary cilia assembly or function are tightly coupled todevelopmental disorders and diseases in mammals termed {"}ciliopathies{"}.The primary objective of this thesis was to investigate the role the primary cilium in coordinating Hh signaling instem cell differentiation and heart development in the mouse. We show that human embryonic stem cells (hESC)and mouse embryonal carcinoma stem cells (P19.CL6 EC cells) have primary cilia that display ciliary localizationof the essential Hh proteins; Gli2, Ptc1 and Smo. Inhibition of the Hh pathway by KAAD-cyclopamine in P19.CL6cells hinder formation of synchronously beating clusters of cardiomyocytes. Knockdown of the primary cilium inP19.CL6 EC cells by nucleofection with plasmids expressing Ift20 and Ift88 siRNA significantly reduced theappearance of beating cardiomyocyte clusters thereby mimicking the effect of cyclopamine treatment. In vivoexperiments revealed that mouse E11.5 Ift88-/- null mutants (which have no primary cilia) have severeendocardial cushion defects, decreased trabeculation and increased pericardial space along with shortened andmalformed cardiac outflow tract. These observations suggest that primary cilia coordinate Hh signaling in stemcell differentiation and cardiogenesis. In support of this, preliminary chimera mouse studies showed thatprimary cilia are important for heart development. This was judged by the distribution of enzymatically taggedwt and Ift88-/- ES cells in the developing heart at E8.5, where only the wt cells are localized to the heartchambers. This signifies that primary cilia are needed for the formation of the heart chambers.The secondary thesis objective was to investigate the role of progesterone signaling in the female reproductionorgans in addition to the role of primary cilia in human pancreatic development and cancer. The findings of theprogesterone receptor in the lower half of the motile cilia in the oviduct, suggest a sensory role of motile cilia inprogesterone signaling where they might coordinate post ovulatory events. In tissue sections of the developinghuman pancreas we found up to 20µm long primary cilia projecting into the duct lumen of the exocrine duct,which have increased ciliary localization of Gli2 and Smo after initiation of fetal development, i.e., at weeks 14and 18. In contrast, ciliary localization of these Hh components was absent at the embryonic stage ofdevelopment, i.e., at week 7.5. This suggests a role of primary cilia in coordinating Hh signaling in humanpancreatic development and postnatal tissue homeostasis. In cultures of human pancreatic duct adenocarcinomacell lines PANC-1 and CFPAC-1, Ptc in addition to Gli2 and Smo localize to primary cilia. These findings areconsistent with the idea that the primary cilium continues to coordinate Hh signaling in cells derived from themature pancreas. The fact that the Hh signaling pathway is active in the CFPAC-1 and PANC-1 cell lines withoutHh stimulation suggests that ciliary Hh signaling plays a potential role in tumorigenesis.In conclusion, this thesis supports the idea that both motile and primary cilia are critical organelles in thecoordination of developmental processes and tissue function.",

author = "Clement, {Christian Alexandro}",

note = "Academic advisor: S{\o}ren Tvorup Christensen",

year = "2009",

month = nov,

day = "15",

language = "English",

publisher = "Museum Tusculanum",

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TY - BOOK

T1 - Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis

AU - Clement, Christian Alexandro

N1 - Academic advisor: Søren Tvorup Christensen

PY - 2009/11/15

Y1 - 2009/11/15

N2 - This thesis focuses on cilia and their sensory function in the mammalian organism. In particular, the Hedgehog(Hh) signaling pathway functions via the primary cilium and plays a unique role in development, differentiation,cancer and possibly in stem cell fate. Defects in primary cilia assembly or function are tightly coupled todevelopmental disorders and diseases in mammals termed "ciliopathies".The primary objective of this thesis was to investigate the role the primary cilium in coordinating Hh signaling instem cell differentiation and heart development in the mouse. We show that human embryonic stem cells (hESC)and mouse embryonal carcinoma stem cells (P19.CL6 EC cells) have primary cilia that display ciliary localizationof the essential Hh proteins; Gli2, Ptc1 and Smo. Inhibition of the Hh pathway by KAAD-cyclopamine in P19.CL6cells hinder formation of synchronously beating clusters of cardiomyocytes. Knockdown of the primary cilium inP19.CL6 EC cells by nucleofection with plasmids expressing Ift20 and Ift88 siRNA significantly reduced theappearance of beating cardiomyocyte clusters thereby mimicking the effect of cyclopamine treatment. In vivoexperiments revealed that mouse E11.5 Ift88-/- null mutants (which have no primary cilia) have severeendocardial cushion defects, decreased trabeculation and increased pericardial space along with shortened andmalformed cardiac outflow tract. These observations suggest that primary cilia coordinate Hh signaling in stemcell differentiation and cardiogenesis. In support of this, preliminary chimera mouse studies showed thatprimary cilia are important for heart development. This was judged by the distribution of enzymatically taggedwt and Ift88-/- ES cells in the developing heart at E8.5, where only the wt cells are localized to the heartchambers. This signifies that primary cilia are needed for the formation of the heart chambers.The secondary thesis objective was to investigate the role of progesterone signaling in the female reproductionorgans in addition to the role of primary cilia in human pancreatic development and cancer. The findings of theprogesterone receptor in the lower half of the motile cilia in the oviduct, suggest a sensory role of motile cilia inprogesterone signaling where they might coordinate post ovulatory events. In tissue sections of the developinghuman pancreas we found up to 20µm long primary cilia projecting into the duct lumen of the exocrine duct,which have increased ciliary localization of Gli2 and Smo after initiation of fetal development, i.e., at weeks 14and 18. In contrast, ciliary localization of these Hh components was absent at the embryonic stage ofdevelopment, i.e., at week 7.5. This suggests a role of primary cilia in coordinating Hh signaling in humanpancreatic development and postnatal tissue homeostasis. In cultures of human pancreatic duct adenocarcinomacell lines PANC-1 and CFPAC-1, Ptc in addition to Gli2 and Smo localize to primary cilia. These findings areconsistent with the idea that the primary cilium continues to coordinate Hh signaling in cells derived from themature pancreas. The fact that the Hh signaling pathway is active in the CFPAC-1 and PANC-1 cell lines withoutHh stimulation suggests that ciliary Hh signaling plays a potential role in tumorigenesis.In conclusion, this thesis supports the idea that both motile and primary cilia are critical organelles in thecoordination of developmental processes and tissue function.

AB - This thesis focuses on cilia and their sensory function in the mammalian organism. In particular, the Hedgehog(Hh) signaling pathway functions via the primary cilium and plays a unique role in development, differentiation,cancer and possibly in stem cell fate. Defects in primary cilia assembly or function are tightly coupled todevelopmental disorders and diseases in mammals termed "ciliopathies".The primary objective of this thesis was to investigate the role the primary cilium in coordinating Hh signaling instem cell differentiation and heart development in the mouse. We show that human embryonic stem cells (hESC)and mouse embryonal carcinoma stem cells (P19.CL6 EC cells) have primary cilia that display ciliary localizationof the essential Hh proteins; Gli2, Ptc1 and Smo. Inhibition of the Hh pathway by KAAD-cyclopamine in P19.CL6cells hinder formation of synchronously beating clusters of cardiomyocytes. Knockdown of the primary cilium inP19.CL6 EC cells by nucleofection with plasmids expressing Ift20 and Ift88 siRNA significantly reduced theappearance of beating cardiomyocyte clusters thereby mimicking the effect of cyclopamine treatment. In vivoexperiments revealed that mouse E11.5 Ift88-/- null mutants (which have no primary cilia) have severeendocardial cushion defects, decreased trabeculation and increased pericardial space along with shortened andmalformed cardiac outflow tract. These observations suggest that primary cilia coordinate Hh signaling in stemcell differentiation and cardiogenesis. In support of this, preliminary chimera mouse studies showed thatprimary cilia are important for heart development. This was judged by the distribution of enzymatically taggedwt and Ift88-/- ES cells in the developing heart at E8.5, where only the wt cells are localized to the heartchambers. This signifies that primary cilia are needed for the formation of the heart chambers.The secondary thesis objective was to investigate the role of progesterone signaling in the female reproductionorgans in addition to the role of primary cilia in human pancreatic development and cancer. The findings of theprogesterone receptor in the lower half of the motile cilia in the oviduct, suggest a sensory role of motile cilia inprogesterone signaling where they might coordinate post ovulatory events. In tissue sections of the developinghuman pancreas we found up to 20µm long primary cilia projecting into the duct lumen of the exocrine duct,which have increased ciliary localization of Gli2 and Smo after initiation of fetal development, i.e., at weeks 14and 18. In contrast, ciliary localization of these Hh components was absent at the embryonic stage ofdevelopment, i.e., at week 7.5. This suggests a role of primary cilia in coordinating Hh signaling in humanpancreatic development and postnatal tissue homeostasis. In cultures of human pancreatic duct adenocarcinomacell lines PANC-1 and CFPAC-1, Ptc in addition to Gli2 and Smo localize to primary cilia. These findings areconsistent with the idea that the primary cilium continues to coordinate Hh signaling in cells derived from themature pancreas. The fact that the Hh signaling pathway is active in the CFPAC-1 and PANC-1 cell lines withoutHh stimulation suggests that ciliary Hh signaling plays a potential role in tumorigenesis.In conclusion, this thesis supports the idea that both motile and primary cilia are critical organelles in thecoordination of developmental processes and tissue function.

M3 - Ph.D. thesis

BT - Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis

PB - Museum Tusculanum

CY - Biologisk Institut

ER -

Primary cilia and coordination of signaling pathways in heart development and tissue Homeostasis

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