Apical Ectodermal Ridge (AER)

190 Pages·2010·8.53 MB·English

by  Ana Raquel Tomás

Apical Ectodermal Ridge (AER) activity
and Limb outgrowth during
Vertebrate Development

Ana Raquel Viegas Tomás

TESI DOCTORAL UPF
2010

Thesis Director: Joaquín Rodríguez León

Centre de Medicina Regenerativa de Barcelona (CMRB) /
Facultad de Medicina de la Universidade de Extremadura (UNEX)

Aos meus Pais…

AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT

TABLE OF CONTENTS
Acknowledgements...................................................................................................ix 
List of Abreviations..................................................................................................xi 
Summary.................................................................................................................xiii 
Resumen.....................................................................................................................xv 
INTRODUCTION......................................................................................................1 
I  Overview of vertebrate limb development..........................................................3 
I.1  Proximodistal limb bud development...............................................................5 
I.2  Limb bud positioning, initiation and early outgrowth......................................5 
I.2.1  Establishment and maintenance of the fgf8/fgf10 positive feedback loop....7 
I.2.2  Retinoic acid and the promotion of Proximal-Distal (PD) outgrowth..........9 
I.3  Establishment of dorso-ventral (DV) polarity and patterning........................11 
I.4  ZPA and the regulation of Anterior-Posterior (AP) patterning......................11 
I.5  Axes coordination during limb development.................................................14 
I.5.1  Proposed models for axes coordination during limb development.............15 
II  Developmental dynamics of the AER..............................................................17 
III  FGF signalling pathway regulation................................................................20 
III.1  Ras-MAPK/ERK pathway...........................................................................20 
III.2  PI3 kinase/Akt pathway...............................................................................21 
III.3  Modulators of FGF signalling.....................................................................22 
III.3.1  Negative regulators: sef, sprouty and mkp3...............................................23 
III.3.2  Positive regulators: flrt3.............................................................................24 
IV  The AER as a model for the study of epithelial renewal................................26 
IV.1.1  The stemness marker, Oct4........................................................................28 
AIMS..........................................................................................................................31 
RESULTS..................................................................................................................33 
I  Flrt3 as a key player in Limb Development......................................................37 
Overview.................................................................................................................37 
I.1  FLRT3 phylogenetic analysis.........................................................................39 
I.2  Danio rerio’s flrt3..........................................................................................40 
I.2.1  Gene expression pattern of flrt3 during zebrafish development.................42 
I.2.2  Immunolocalization of FLRT3 during pectoral fin development...............42 
I.3  Gallus gallus’s flrt3........................................................................................43 
I.3.1  Flrt3 expression is restricted to the AER and coincides with that of fgf8 and
pERK activity..........................................................................................................43 
I.3.2  Immunolocalization of FLRT3 during limb development..........................46 
I.3.3  Overexpression of flrt3 in the limb ectoderm induces ectopic ridges and
enlargements of the pre-existent AER....................................................................47 

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A.R.TOMÁS, 2010 TABLE OF CONTENTS
AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT
I.3.4  Silencing of flrt3 affects the integrity of the AER.....................................52 
I.3.5  Flrt3 expression is not regulated by FGF activity, although ectopic Wnt3a
is able to induce flrt3..............................................................................................55 
I.3.6  BMPs specifically inhibit flrt3 in the AER................................................57 
I.4  Gallus gallus’s flrt2.......................................................................................58 
II  Oct4 sustains Apical Ectodermal Rigde renewal............................................61 
Overview................................................................................................................61 
II.1  Oct4 is expressed during limb development................................................63 
II.2  Cell dynamics in the AER............................................................................64 
II.3  Oct4 overexpression expands the AER and enlarges the limb mesenchyme67 
II.4  AER cell dynamics upon oct4 overexpression is altered.............................69 
II.5  FGF signalling is needed to maintain oct4 expression.................................71 
II.6  The canonical WNT, WNT3A, controls oct4 expression in the AER.........74 
II.7  BMPs negatively control oct4 expression prior to induction of cell death..74 
II.8  RA activity controls oct4 expression but cannot induce an ectopic AER....75 
II.9  Other candidate genes for oct4 co-regulation during AER renewal............75 
II.9.1  Retinoic acid metabolic enzymes, cyp26 and raldh2................................76 
II.9.2  Sox14.........................................................................................................77 
II.9.3  p27Kip1........................................................................................................78 
II.9.4  Lgr5...........................................................................................................78 
DISCUSSION...........................................................................................................81 
I  Flrt3 and vertebrate limb development............................................................83 
I.1  Flrt3 is essential for AER integrity and activity...........................................84 
I.2  Flrt3 is necessary but not sufficient for proper AER formation and
maintenance............................................................................................................85 
I.3  Limb territorial borders are shiffted but identities are not altered upon Flrt3
overexpression in the limb ectoderm.....................................................................86 
I.4  Flrt3 expression: the effect of FGF and WNT signalling.............................87 
I.5  Flrt3 regulate AER’s BMP signalling through gremlin................................88 
I.6  Proposed model for flrt3 action in chick limb development.........................88 
I.7  Flrt3 in zebrafish fin development................................................................89 
II  Flrt3 in other FGF signalling centers..............................................................91 
III  Flrt2...............................................................................................................93 
IV  AER cell dynamics........................................................................................94 
IV.1  Other epithelial cell renewal systems: the small intestine..........................95 
V  Oct4 in AER development and renewal..........................................................96 
V.1  RA and BMPs negativelly regulate oct4......................................................97 
V.2  Oct4 is upregulated through exogenous application of FGFs and WNT3A 99 
VI  Oct4 co-regulators of AER renewal............................................................101 
VI.1  Sox14 and p27, onsets for AER cell differentiation?................................101 
VI.2  Lgr5..........................................................................................................103 
VII  Proposed model for AER activity and renewal and involved players........105 
CONCLUSIONS....................................................................................................107 

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AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT
MATERIALS AND METHODS...........................................................................111 
I  Embryo Models...............................................................................................113 
I.1  Staging, collection and processing of the biological material......................113 
II  Cloning of full-length and dsRNA constructs................................................114 
II.1.1  Full-length flrt3........................................................................................114 
II.1.2  Full-length oct4........................................................................................114 
II.1.3  dsRNA against flrt3.................................................................................115 
III  Chicken Embryo Manipulation.....................................................................116 
III.1  Bead implantation......................................................................................116 
III.2  Electroporation of limb ectoderm..............................................................117 
IV  In situ hybridisation......................................................................................118 
IV.1  Probes........................................................................................................118 
IV.1.1  Cloning of the zebrafish flrt3 probe.........................................................119 
IV.1.2  Cloning of the chicken oct4 probe...........................................................119 
IV.2  Protocols....................................................................................................119 
V  Cryopreservation and sectioning...................................................................120 
VI  Immunohistochemistry.................................................................................120 
VII  Proliferation assays.....................................................................................121 
VII.1  Phospho-Histone H3................................................................................121 
VII.2  BrdU incorporation and detection............................................................121 
VIII  Cell Death assays.......................................................................................122 
VIII.1  In sections...............................................................................................122 
VIII.2  Wholemount............................................................................................123 
IX  Imaging.........................................................................................................123 
X  Alcian green cartilage staining......................................................................123 
XI  Scanning Electron Microscopy.....................................................................124 
REFERENCES.......................................................................................................125 
INDEX of figures....................................................................................................143 
INDEX of tables......................................................................................................151 
ANNEXES...............................................................................................................153 
I  Supplementary data.........................................................................................155 
II  Embryonic stages of the animal models used................................................157 
II.1  Gallus gallus...............................................................................................157 
II.2  Danio rerio..................................................................................................158 
III  Detailed Protocols.........................................................................................159 
III.1  Wholemount in situ hybridisation protocol for chicken embryos.............159 
III.2  Wholemount in situ hybridisation protocol for zebrafish embryos...........161 
III.3  Immunohistochemistry protocol for wholemount zebrafish embryos.......162 
III.4  RNAi cloning.............................................................................................163 
III.5  Production of RCAS virus.........................................................................165 
IV  Sequences.....................................................................................................167 
V  Plasmids vectors............................................................................................171 

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ACKNOWLEDGEMENTS

I would like to acknowledge the funding from Fundação para a Ciência e Tecnologia (SFRH /
BD / 32346 / 2006) and from Fundação Calouste Gulbenkian, that allowed me to complete
the work presented in this thesis.

I would like to acknowledge the institutions where this work has been developed, for having
accepted me as a PhD student at their labs: to the Centre de Medicina Regenerativa de
Barcelona at the Parc de Recerca Biomedica de Barcelona; and to the Departamento de
Anatomía, Biología Celular y Zoología de la Facultad de Medicina da Universidad de
Extremadura at Badajoz. And also to Instituto Gulbenkian de Ciência, that has always been,
and will be, my “home”.

"O caminho faz-se caminhando."
At the end of this road, I cannot move on without thanking to all the people that came along
on my journey, or just cross my path along these years. This thesis has also a bit of them,
since one way or another, I have grown because of, and thanks to, them... you.

First of all, I would like to thank to Joaquín Rodríguez. Not in a million years I will find a
BOSS like you! As my mother would say: "Nem com uma candeia acessa...!"
I cannot find the words to begin to thank you... maybe I'll just do a cheesecake and we leave it
like this... ; Now seriously, thank you for every tip, every advice, for sharing your knowledge
and for showing me how to proper science! For the opportunity to work in fancy places with
"heavy machinery" and in the smaller ones, and teaching me that you can do great science in
both places (you just have less cultural opportunities available...)
For your friendship, THANK YOU, BOSS!

At the CMRB, a big thank to you all for accepting “la portuguesa” with arms wide open,
especially to Adriana and Dani (gracias, amigos! Demasiadas horas cerrados en la “chicken”
han hecho esto - que jamás me olvide de vosotros); to my fellow “becarios” at CMRB, the
postdocs and amazing technical staff, and administrative staff, that I cannot name all because
they are too many… Thank you.

At UNEX, A Yolanda y Domingo. Os agradezco muchissimo. A todo el personal en el
departamento por hacer com que me sienta “en casa”. Gracias!

At IGC, Catarina (sem palavras, obrigada!), um obrigado colectivo a todos os Organogénicos:
um obrigado por tudo, in situs e caipirinhas, tudo faz parte do pacote! Agradeço em pessoa e
ao vivo!

Surely there are a lot more people to thank, but time and space contingencies do not allow
me.

Aos meus pais, que sempre me apoiaram nestas minhas “aventuras” não consigo passar a
palavras o quanto eu vos estou grata!

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A.R.TOMÁS, 2010 ACKNOWLEDGEMENTS
AER ACTIVITY AND LIMB OUTGROWTH DURING VERTEBRATE DEVELOPMENT

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A.R.TOMÁS, 2010 ACKNOWLEDGEMENTS