Long-range signalling of TGF\03B2 morphogens in the early Xenopus embryo

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Universidade de Lisboa

Faculdade de Ciências de Lisboa

Departamento de Biologia Animal

Long-range signalling of TGFβ morphogens in the

early Xenopus embryo

Anja Irene Hagemann

Doutoramento em Biologia

(Biologia do Desenvolvimento)

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Universidade de Lisboa

Faculdade de Ciências de Lisboa

Departamento de Biologia Animal

Long-range signalling of TGFβ morphogens

in the early

Xenopus

embryo

Anja Irene Hagemann

Tese orientada por:

Professora Doutora Sólveig Thorsteinsdóttir

e Professor Doutor Jim C. Smith

Doutoramento em Biologia

(Biologia do Desenvolvimento)

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Os trabalhos apresentados nesta tese foram realizados com o apoio financeiro da Fundação para a

Ciência e a Tecnologia (bolsa de referência SRFH/BD/11807/2003) e, posteriormente, da Fundação

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NOTAS PRÉVIAS

1) Para a elaboração da presente tese de doutoramento foram usados integralmente como capítulos artigos científicos publicados, ou submetidos para publicação, em revistas científicas internacionais indexadas. Uma vez que estes trabalhos foram realizados em colaboração com outros investigadores, e de acordo com o disposto no nº 1 do Artigo 41º do Regulamento de Estudos Pós-Graduados da Universidade de Lisboa, publicado no Diário da República, 2.a_{série, n.}o_{209, 30 de Outubro de 2006, esclareço que participei integralmente na}

concepção e execução do trabalho experimental, na interpretação dos resultados e na redacção dos manuscritos. Excepcionalmente, no artigo apresentado no Capítulo 2, secção 2.1. desta tese, “Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo” (Current Biology 14 (2004), 1916-1923), a minha contribuição é restringida aos resultados referentes às figuras 3B-I, 4A’ e 5D. No artigo apresentado no Capítulo 2, secção 2.2. desta tese, “Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus” (Development 134 (2007), 4209-4218), os resultados referentes às figuras 1, 2A, 3A/B e à tabela 1 foram integralmente obtidos por Yasushi Saka. No artigo apresentado no Capítulo 2, secção 2.3. desta tese, “Visualizing protein interaction by bimolecular fluorescence complementation in Xenopus.” a minha contribuição é restringida aos protocolos da microscopia confocal e à figura 2.

2) O facto de esta tese integrar vários artigos científicos levou a que a redacção dos vários capítulos tenha sido feita de acordo com as normas de cada revista, variando, portanto, ao longo desta tese. O Capítulo 2.3 consta de um artigo submetido para publicação e encontra-se formatado de acordo com as regras exigidas pela revista Current Biology.

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ACKNOWLEDGEMENTS

The last years were probably the most exciting of my life. Some times have been rather comparable to a long trip in a roller coaster and had I not had such caring and inspiring people around me all the time, I might not be where I am now. So, its time to thank everyone involved in making this a great experience. Since one reason for these years being so exciting has been the immense number of people I met, I want to apologize in advance to those that I might have forgotten in the following paragraphs.

Firstly I want to thank Antonio Coutinho for convincing me to join the Programma Gulbenkian Doutoramento em Biomedicina (PGDB), probably one of the best PhD programmes, that changed my life so fundamentally and that sadly doesn’t exist anymore. Many thanks to Sukalyan Chatterjee for both the guidance and the freedom he gave his students and the great organization of the first year with many inspiring teachers. I want to thank him and Miguel Seabra for their permission for me to do the practical part of my dissertation here in Cambridge, UK. In this context I also want to thank Solveig Torsteinsdottir and in particular Antonio Jacinto for their help throughout my PhD, and their support when I decided to change project and laboratory.

There is no doubt that I could not have completed this PhD without one of the best supervisors, Jim Smith, who is responsible for me growing from a technician into a scientist. His inspiring discussions and cheer leader attitude were at many times vital for the success of my project, even providing friendship when needed. Thank you!

The whole Smith-lab was a terrific place to work with a great bunch of individuals. I want to thank all past and present members, particularly Kevin Dingwell and Oli Nentwich for scientific discussions and their help in the lab particularly as I was new to working with frogs; Clara Collart, Mike Chesney and Amer Rana for many fruitful discussions, science related or not; Amanda Evans, Kim Lachani, Kevin Dingwell, Nigel Messenger, David Simpson and Katie Woodhouse for their irreplaceable management of the lab and frog facility; Julia Bates for keeping order where there was chaos; Yasushi Saka, Xin Xu and Oliver Nentwich for fruitful collaborations; Joanna Argasinska, Dunja Knapp, and Joana Ramis for lots of laughs. Especially Oli, Xin, Joanna and Liz have been really friends in good and bad times. Thank you for brightening my days!

I obviously could not write this chapter without mentioning the numerous coffees I enjoyed with Liz, exchanging gossip, many motivating science discussions and loads of laugh-muscle training.

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It might seem out dated, but I cannot deny the great help of Alexandre Raposo, an excellent teacher of the Portuguese language with whom I experienced one of the most fun years of my life in Lisbon and who meant comfort and home to me for more than three years here in Cambridge. I learned to know heaven and hell with you.

I want to thank all members of the PGDB3 who I will always remember as the “positive crowd” with whom I had such a great time in Lisbon and the Gulbenkian Institute. Particularly Alexandra Pacheco, and Rita Silva have become good friends. I thank the hard core of the Wednesday wine loving “Schnell-friends” as there are Hatice Akarsu, Mike Chesney and Tristana von Will (VIS), Karin Edoff, Harold Aeytey, Petra Heikova, Erna Mágnusdóttir and Sean Jeffries, with whom I enjoyed one or the other glass and debate. Special thanks also to the badminton crew that helped to keep me fit, Bedra, Petra, Karin, Maria-Joao and Hatice; and particularly Anja Hagting, who is responsible for the excellent organisation of the weekly exercise. I also want to thank the former Mill-Roadies, Richard Southern and Daniel Lackner for delicious dinners; and for regular popcorn supply I thank Caroline Gasparin, who I always admired for her endurance to sit on a chair.

A very big thank you goes to Solveig Torsteinsdottir, Fernanda Bajanca, Maria-Joao Amorim, Ricardo Costa, Liz Callery and Mike Chesney for very helpful comments on the manuscript. A very special friend needs to be mentioned that somehow always managed to show me the bright side of life, my little sister Hatice, twin in spirit for the depths and joys of life. I also want to thank a very good friend, Francesca Cesari, one of the best listeners with whom I shared ups and downs during the whole time I have been in Cambridge. I shall not forget Moises Mallo, a friend for a lifetime, for his daily-(e-)mail and all his support. All these friends kept my spirit on the bright side of life.

I want to thank my dad, Erika and my step sisters, Kathrin and Angelika for their support and the understanding that during this overwhelming time I haven’t been in contact as much as I should have.

I would not be who I am and where I am without my mother, who I thank for always believing in me, for supporting me in all my decisions, for teaching me how wonderful nature is and to whom I want to dedicate this dissertation.

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RESUMO

Este estudo teve como objectivo elucidar a função que os factores transformantes e de crescimento tipo beta (Transforming growth factor type beta; TGFβ) exercem durante o desenvolvimento da mesoderme em Xenopus laevis e o processo pelo qual esses mesmos morfogénios se movem através de um campo composto por um conjunto de células. A estratégia utilizada foi a construção de proteínas recombinantes de morfogénios marcados com fluorescência de forma a permitir a sua visualização e a análise da sua função. Esta análise foi complementada pelo método de complementação bi-molecular de fluorescência (BiFC) que permite analisar a dinâmica do complexo Smad resultante da via de transdução de sinal TGFβ. A combinação de ambas as técnicas de imagem permitiu observar o comportamento do morfogénio e da sua resposta celular directa .

O modo de funcionamento dos morfogénios é de grande interesse para a biologia de desenvolvimento e medicina porque é um processo essencial que, se interrompido durante a embriogénese, leva a fenótipos graves e quase sempre letais. Assim, não é surpreendente que este processo seja bastante complexo e que tenha sido foco de diversos estudos ao longo das últimas décadas. Nesta tese utilizamos proteínas da família TGFβ para ilustrar a acção de um morfogénio no início do desenvolvimento de Xenopus laevis, em particular durante a indução da mesoderme e a subdivisão da mesoderme em diferentes domínios.

Em 1969, Lewis Wolpert descreveu os aspectos da acção de morfogénios no seu modelo da bandeira Francesa (French flag model). De acordo com o seu modelo, um morfogénio tem de satisfazer dois critérios: (1) ser capaz de actuar à distância e (2) induzir respostas diferentes em função da sua concentração. Segundo a sua teoria, a formação de um gradiente de um morfogénio será o único fenómeno necessário para obter diferentes respostas celulares num conjunto de células. Para a formação desse gradiente, será necessário uma fonte, donde o morfogénio solúvel se difundirá ao longo do campo de células, e um local onde este é absorvido, formando-se assim um gradiente. Em termos práticos, o local de produção poderia ser uma área definida por células secretoras do morfogénio, como por exemplo as células no pólo vegetal da blástula de Xenopus. O conceito de absorção contudo será mais difícil definir, presumindo que nenhuma proteína se perde a partir do epitélio no pólo animal da blástula de Xenopus. Contudo, é possível atingir um efeito de gradiente através da constante internalização do morfogénio por um receptor celular e/ou da sua degradação ao longo do trajecto. Desde que as células sejam sensíveis à concentração do morfogénio, estas responderão de diferente forma. É esta situação que está explícita no modelo da bandeira

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francesa do Wolpert; onde ele ilustra as diferentes respostas à concentração de um morfogénio com a expressão de cores diferentes. Células mais próximas produzem uma tonalidade azul em função da maior concentração do morfogénio, células numa posição intermédia apresentam uma cor branca, e células ainda mais distantes, aquelas que recebem menos morfogénio, apresentam uma cor vermelha. O resultado é um padrão às riscas semelhante à bandeira francesa.

A mesoderme do embrião de Xenopus forma-se a partir da região equatorial da blástula em resposta a sinais provenientes do hemisfério vegetal do embrião. Estes sinais incluem membros da família de TGFβ, incluindo Vg1, activin, proteínas relacionadas com nodal (Nodal related proteins; Xnr1, 2, 4, 5 e 6) e derrière. Eu escolhi activin como representante de proteínas da família TGFβ indutoras de mesoderme para experiências sobre a progressão de morfogénios num campo de células e a transdução de sinal necessária à formação e subdivisão da mesoderme. A activin induz a diferenciação da mesoderme dorsal, assim como movimentos celulares característicos desse tecido, como por exemplo o elongamento em explantes. Estes movimentos estão directamente relacionados com os movimentos morfogenéticos de extensão convergente que ocorrem na mesoderme dorsal em embriões. É bem conhecido que a diferenciação de diferentes tipos de tecidos ao longo do eixo antero-posterior do embrião induzida pela proteína activin é dependente da sua concentração. Explantes do pólo animal da blástula de anfíbios ou “animal caps” diferenciam tanto em epiderme como em tecido mesênquimatoso na presença de baixas concentrações da proteína activin. As mesmas ”animal caps” diferenciam-se em tecido muscular e nervoso em presença de concentrações intermédias de activin, notochorda e desenvolvimento neuronal ocorre na presença de elevadas concentrações de activin.

Nesta tese, investigamos a progressão de activin e Xnrs durante as primeiras fases de desenvolvimento do embrião de Xenopus. Durante este estudo foi analisada de uma forma directa a distribuição e o movimento destes morfogénios in vivo. Numa tentativa de visualizar os ligandos da família TGFβ em tempo real, estas proteínas foram ligadas a uma proteína fluorescente e a sua funcionalidade testada. Numa colaboração com Huw Williams a proteína recombinante EGFP-Xnr2 originou uma proteína funcional (Capítulo 2.1) e, através de um ensaio "fibro-cap" envolvendo hibridação RNA:RNA in situ, demonstramos que EGFP-Xnr2 tem actividade à distância. Neste ensaio “fibro-cap” duas “animal caps” (explantes do pólo animal) aderem a uma superfície revestida por fibronectina que as mantêm esticadas e juxtapostas, desta forma servindo como dador e receptor de transmissão de sinal à distância, respectivamente. Nestas experiências a visualização da distribuição de EGFP-Xnr2 mostrou ser extracelular (Capítulo 2.1)

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A maioria dos estudos com morfogénios aproveitam o facto que há indução de expressão de genes diferentes dependente da concentração do morfogénio em questão. Porém, visto que muitos destes genes são regulados por feedback positivo ou negativo, a interpretação dos resultados pode ser difícil. Numa tentativa de resolver esta limitação técnica, desenvolvemos um método que permite a visualização directa da resposta celular à transdução de sinal TGFβ (Capítulo 2.2.).

O primeiro passo resultante da activação da transdução de sinal por ligandos TGFβ é a fosforilação pelos receptores de R-Smads (Smad2) que pode ser detectada com anticorpos específicos em tecidos fixos. Assim sendo, adoptamos a técnica desenvolvida para a detecção de interacção proteína-proteina em tempo real para observar as interacções dos complexos de Smad em resposta à via de transdução de sinal por TGFβ em embriões de Xenopus (Capítulo 2.2.). O método de complementação bi-molecular de fluorescência (BiFC) foi desenvolvido para estudar interacções proteína-proteina in vivo e foi já utilizado numa variedade de proteínas e em vários tipos de células e organismos. A análise das interacções de proteínas por BiFC baseia-se na formação de um complexo fluorescente aquando da união de dois fragmentos complementares que estão ligados a duas outras proteínas de interesse, sendo que isoladamente esses fragmentos não apresentam fluorescência.

A fluorescência pode ser detectada devido a interacção in vivo das duas proteínas de interesse, cada uma delas ligada a um fragmento do fluoróforo. BiFC permite visualizar a interacção de R-Smad activo com Co-Smad4 e a sua acumulação no núcleo em resposta à transdução de sinal de TGFβ. Este método altamente sensível permitiu desenvolver uma análise quantitativa da resposta à distância de morfogénios TGFβ. Além disso, a aplicação deste método a células embrionárias elucidou detalhes surpreendentes acerca da transdução de sinais TGFβ (Capítulo 2.2). Por exemplo, descobrimos que a importação nuclear do complexo Smad é controlado durante o desenvolvimento e só acontece depois do início da transcrição no zigoto. Outras observações resultantes da utilização deste método incluem a co-localização do complexo Smad com a cromatina durante a mitose, que difere do previamente observado para Smad2 isoladamente (Capítulo 2.2). As nossas experiências sugerem a existência de um mecanismo inesperado que controla a iniciação de transcrição no “midblastula stage” de Xenopus. BiFC forneceu então uma forma directa, rápida e quantitativa para avaliar a transdução do sinal de proteínas semelhantes a activin.

Com base nestes resultados decidimos aprofundar a questão inicial e utilizar esta técnica para avaliar a transdução do sinal à distância dos membros da família de TGFβ. Para tal injectamos RNA de activin, Xnr1 ou Xnr2 numa “animal cap” e os recombinantes de Smad 2 e Smad 4 BiFC (Smad-BiFC) noutra “animal cap”. Utilizando microscopia confocal e visualizando

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células in vivo, avaliámos se a via de transdução de sinal de Smad tinha sido activada nos explantes adjacentes aos que expressam o morfogénio observada pela acumulação de Smad-BiFC no núcleo e utilizando o método “fibro-cap” acima descrito (Capítulo 2.4.).

Enquanto que a expressão de Xnr2 não induziu a activação de Smad-BiFC acima dos níveis apresentados no controlo, a presença de activin ou Xnr1 resultou numa acumulação de fluorescência numa forma de gradiente no núcleo de células distantes das células sinalizadoras que secretam o morfogénio. A taxa de activação de Smad-BiFC, medida em termos da sua acumulação no núcleo, está directamente correlacionada com a quantidade de morfogénio e apresentou-se estável após uma hora e meia de experiência, o que sugere que o método de Smad-BiFC “fibro-cap” é rápido, directo e permite detectar a transdução de sinais TGFβ à distância (Capítulo 2.4.).

Num esforço para visualizar a transmissão de sinal à distância em conjunto com a distribuição do morfogénio, marcamos diversas proteínas TGFβ com GFP. Infelizmente, a proteína activin ou Xnr1 marcada com uma proteína fluorescente originou uma proteína recombinante pouco funcional. No entanto, uma colaboração com Marko Hyvonen, permitiu-nos marcar a proteína activin com o fluoróforo Alexa488, e após a realização de uma série de experiências conseguimos determinar que a activin-alexa488 retêm 50% da actividade original (Capítulo 2.4.). O tratamento de células provenientes do pólo animal de Xenopus mostrou que estas células internalizam agregados de activin-alexa488 de uma forma específica. No entanto, de forma a visualizar a passagem da proteína activin em tecidos compactos, introduzimos esferas Affi-gel previamente incubadas com o conjugado activin-alexa488 num dos explantes e adicionamos este terceiro componente ao método de “fibro-cap”,

O método, “triple-cap” permite assim um espaço quase ilimitado para a progressão do ligando. A distribuição de activin-alexa488 em “animal caps” da blástula de Xenopus mostrou um comportamento dinâmico ao longo do tempo. De tal forma que, duas horas após a introdução das esferas, podia detectar-se fluorescência na matriz extracelular (ECM) e em quantidades ínfimas no interior das células. Sendo assim, estes resultados são comparáveis aos resultados obtidos anteriormente com EGFP-Xnr2. No entanto, três horas e meia depois da implantação das esferas Affi-gel, o conjugado activin-alexa488 encontra-se no interior das células mais próximas das esferas e forma agregados que contrasta com a situação anterior em que a fluorescência era difusa (Capítulo 2.4.).

Transcitose de um morfogénio envolve a endocitose de complexos ligando-receptor na membrana citoplasmática, seguido pela sua reciclagem através do lúmen celular de forma a distanciar-se do seu local de produção. Este mecanismo tem sido proposto como uma das

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formas possíveis pelo qual activin e outros membros da família TGFβ podem exercer os seus efeitos a longas distâncias. Este mecanismo foi inicialmente descrito para o morfogénio dpp (decapentaplegic) no disco imaginal de Drosophila, mas, contrariamente ao epitélio do disco imaginal de Drosophila, o mesênquima da blástula de Xenopus é um tecido organizado de uma forma solta contendo grande espaços extracelulares. Portanto pode facilmente haver espaço para a passagem de proteínas entre as células. No Capítulo 2.4 investigamos se a passagem da proteína activin pode ocorrer desde o lúmen de uma célula para outra, utilizando células de blástulas e gastrulas. Experiênicas onde utilizamos uma mistura de células, algumas das quais tinham internalizado activin e outras que não continham activin sugerem que, contrariamente a outros morfogénios, a formação de gradiente de activin não envolve este método de passagem do ligando de célula para célula e que activin parece viajar exclusivamente pelo espaço extracelular (Capítulo 2.4.).

Outro aspecto que tem sido proposto como capaz de influenciar a passagem de morfogénios é a capacidade de internalização de complexos ligando-receptor por endocitose. Este mecanismo foi proposto como um factor regulador na formação de gradientes de morfogénios, para um certo número de factores tais como fgf8, wnt ou hh. O conceito de que há uma necessidade de endocitose para a transdução de sinais TGFβ no entanto é controverso. Alguns investigadores sugerem que a endocitose é essencial enquanto que outros sugerem que a transdução de sinal desencadeada por TGFβ ou activin pode ocorrer a partir da membrana citoplasmática. Nesta tese testamos o efeito da endocitose na transdução do sinal de activin e na sua resposta à distância. A expressão de uma forma dominante negativa de Rab5 (DNRab5), que bloqueia endocitose, mostra claramente uma inibição da internalização de agregados do conjugado activin-alexa488, quer em células dissociadas de “animal caps” quer em “fibro-caps”. Surpreendentemente, esta inibição da endocitose não resultou em alterações na capacidade de sinalização de activin à distância, observada pela acumulação nuclear de Smad-BiFC em células adjacentes ao tecido secretor de activin. Em colaboração com Xin Xu, investigamos o efeito de DNRab5 na indução de genes alvo em explantes do pólo animal (“animal caps”). A activação de genes que são alvos directos de activin não diminuiu em função da expressão de DNRab5 nesses tecidos, enquanto que em células dissociadas na mesma experiência se pode observar uma diminuição de cerca de 90% na internalização do conjugado activin-alexa488, quando comparado com as células em condições de controlo. A inibição da endocitose permitiu assim uma inibição da internalização de activin, mas essa inibição em nada interferiu com a capacidade de transmissão do sinal à distância. Estas experiências sugerem que a maioria da proteína activin-alexa488 internalizada não contribui para a transmissão do sinal (Capítulo 2.4).

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Seguidamente pretendemos elucidar a função destes agregados nas células e saber qual o compartimento celular onde se localizam. A transdução de sinal via receptores hetero-diméricos TGFβ tipo I ou tipo II, tem sido proposta como ocorrendo através de endossomas revestidos de clatrina, enquanto que a reciclagem do receptores de TGFβ ocorre através de “caveolae”. Investigamos então a co-localização de activin com marcadores específicos para endossomas iniciais e lisossomas para determinar a localização subcelular dos agregados de activin-alexa488. As células foram depois tratadas com activin-alexa488 durante uma hora e processadas por microscopia confocal. Apenas em poucos casos encontrou-se uma localização de activin-alexa488 com Rab5 em endossomas iniciais, enquanto que a co-localização de activin-alexa488 com lisossomas era muito frequente. Este resultado sugere que a activin-alexa488 passa através de endossomas iniciais contendo Rab5 de uma forma muito rápida e é depois enviada para degradação nos lisossomas. É interessante salientar que a inibição da via endocítica dependente de Rab5 parece não ser fundamental quer no processo de sinalização à distância, quer na activação da expressão de genes alvo de activin.

Como forma de conclusão, o conjunto destes resultados levam-nos a propôr que os morfogénios que induzem a formação da mesoderme no embrião de Xenopus viajam exclusivamente no espaço extracelular. A transmissão do sinal de activin à distância e a indução da expressão de genes alvo é independente da endocitose Rab5-dependente. Estes resultados levam-nos também a propôr que a formação dos complexos de Smad são um processo regulado durante o desenvolvimento e podem ter uma função adicional e inesperada durante a divisão celular.

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ABSTRACT

The mesoderm of the amphibian embryo forms from the equatorial region of the late blastula in response to signals derived from the vegetal hemisphere of the embryo. These signals include members of the transforming growth factor type β (TGFβ) family, including Vg1, Activin, the nodal-related proteins Xnr1, 2, 4, 5 and 6, and derrière. Two significant characteristics of these signals are that they can act at long range and that they behave as morphogens, eliciting different responses at different concentrations. These properties raise the question of how such molecules exert their long-range effects: what route do they take to traverse a field of responding cells?

As a representative of mesoderm inducing TGFβ molecules, we have used mainly Activin for our experiments addressing questions about morphogen progression and signalling. Activin is a potent inducer of mesoderm and its patterning. We developed several different assays involving a fluorescently labelled form of the protein to visualize morphogen progression in tissue. In vivo detection of TGFβ induced complex formation of signal transducers such as Smad2 and Smad4 by bimolecular fluorescent complementation (BiFC) served as a direct read-out of morphogen function. The combination of both imaging techniques allowed us to observe morphogen passage and direct response in living cells. Our experiments suggest that in contrast to other morphogens, Activin does not pass through neighbouring cells by transcytosis, but appears to travel exclusively through the extracellular space. The number of cognate receptors presented on cell surfaces is crucial for the range of progression and signalling as it blocks ligand passage if presented in excess. Inhibition of endocytosis successfully blocks cellular uptake of Activin, but does not interfere with either signalling capacity or range.

Furthermore, during our studies on Smad complex formation we made several important observations. Most interestingly, we found that the nuclear import of the Smad complex is developmentally regulated such that nuclear accumulation in response to a TGFβ stimulus does not occur before the start of zygotic transcription, indicating the presence of regulatory controls that impose temporal restrictions upon the nuclear import of Smad signalling complexes. Future elucidation of the molecular nature of these import controls may be particularly relevant to our understanding of the mid-blastula transition, as the onset of zygotic gene regulation may require the import of transcriptional regulators.

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LIST OF ABBREVIATIONS

BiFC Bimolecular Fluorescent Complementation Bmp Bone morphogenetic protein

Dpp Decapentaplegic DNA Deoxyribonucleic Acid ECM Extracellular Matrix

EYFP Enhanced yellow fluorescent protein (e)Fgf (embryonic) Fibroblast growth factor

FRET Fluorescence/Förster resonance energy transfer Gsc goosecoid

GFP Green fluorescent protein HSPG Heparan sulfate glycoprotein

Hh hedgehog Shh Sonic hedgehog

mRNA Messenger Ribonucleic Acid

Smad Vertebrate homologues to the Drosophila “mothers against decapentaplegic” and C. elegans SMA I-Smad Inhibitory Smad

R-Smad Receptor Smad

TGFβ Transforming growth factor type beta Wg Wingless

Wnt Wingless / Integrated family members Xbra Xenopus brachyury

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1.1 Early Xenopus development

1.1.1 Morphological pola risation in earl y development

of the

Xenopus

embryo

Developmental biology attempts to understand the processes that turn an egg into a fully formed organism. One of the most fascinating questions in this context is: how is the embryo patterned? The first cues for the formation of the body plan of the South-African clawed frog, Xenopus laevis, become visible in the unfertilized egg as an animal-vegetal polarization. Heavy yolk granules are distributed in a vegetal to animal gradient, while the darker pigmented animal cortex at the upper side disguises the egg in its natural environment. Other visible markers of an animal-vegetal polarisation include the germinal vesicle, the position of the nucleus, in the animal hemisphere and the mitochondrial cloud in the vegetal hemisphere (Figure 1).

Figure 1: Timeline of intracellular organization of the Xenopus egg before and after fertilisation. (A) Anatomy

of the polarized egg with pigmented animal hemisphere, represented as sagittal section. (B) Distribution of maternal VegT, Vg1 and Wnt11 RNAs in the egg. VegT RNA is localized in the vegetal hemisphere and is translated after fertilization. Vg1 RNA is localized in the vegetal cortex of the fully grown oocyte and moves into the vegetal cytoplasm before fertilisation. Wnt11 RNA is tightly localized to the vegetal cortex and shifted towards the future dorsal side as a consequence of fertilisation. (C) Sperm entry in the dorsal hemisphere initiates cortical rotation including localized RNAs.

Fertilisation initiates a reorganisation of the cytoplasm that leads to the definition of the future dorso-ventral axis of the embryo. The sperm entry point defines the future ventral side by initiating a 30° rotation of the cytoplasm relative to the cell cortex. This shift of cortex versus cytoplasm is caused by a microtubule driven reorganisation of the cytoskeleton, and is sometimes visible (especially in newt embryos) as a grey crescent at the presumptive dorsal margin (Figure 1).

Once the animal-vegetal and dorso-ventral axes have been established, cell division begins. The first cell division separates the left side from the right side of the embryo and the second

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separates the ventral from the dorsal half of the egg. The third embryonic cell division is horizontal and divides the embryo into four smaller animal pole cells and four larger vegetal blastomeres. At the start of zygotic transcription, the egg has completed 12 rounds of cell divisions and appears a radially almost symmetric ball of 4096 cells. The blastula has still the same size and shape as the oocyte, because up to the time of feeding, there is no cell growth in the embryo (Nieuwkoop, 1956).

Figure 2: Tissue movements during gastrulation of Xenopus. In the late blastula the mesoderm (red) is located in

the marginal zone, overlaid by a layer of endodermal cells. Gastrulation is initiated by the formation of bottle cells in the blastopore region, which is followed by the involution over the dorsal lip of the blastopore. The marginal zone endoderm, which was on the surface of the blastula, now lies ventral to the mesoderm and forms the roof of the archenteron. At the same time, the ectoderm of the animal cap spreads downward. The mesoderm converges and extends along the antero-posterior axis. The region of involution spreads ventrally to include more endoderm, and forms a circle around a plug of yolky vegetal cells. The ectoderm spreads by epiboly (After Balinsky B. I., 1975).

At the onset of gastrulation, the first embryonic cell shape changes occur when a small group of cells elongate and constrict their distal ends at the future dorsal side of the embryo. These so-called bottle cells are visible as a condensed area of pigmentation and initiate the invagination of dorsal tissue from the marginal zone. Outer layers of mesodermal and endodermal cells involute and then travel in the reverse direction along the blastocoel-roof of ectodermal cell-layers. Involution then progresses to lateral areas and finally to the ventral side of the embryo until all mesodermal and endodermal tissue is moved to the inside of the embryo (Figure 2). The convergence and extension of lateral and ventral tissues towards the dorsal midline finally leads to an elongation of the antero-posterior body axis.

1.1.2 Molecular contro l of axis fo rmatio n of the

Xenopus

embryo

The visible events that occur during early frog development are driven by processes at the molecular level. The established body axes in the oocyte are reflected by the asymmetric

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distribution of a number of maternally provided RNAs encoding proteins like Wnt11, a member of the Wnt family of secreted lipoglycoproteins, dishevelled, a transducer of the canonical Wnt signalling pathway, Vg1, a member of the transforming growth factor superfamily of secreted proteins, and VegT, a T-box transcription factor. All of these mRNAs are tightly concentrated at the vegetal cortex (Figure 1). A smaller number of RNA molecules are localised at the animal side in a more diffuse fashion (Birsoy et al., 2006; Heasman, 2006). Many of these molecules function in patterning the body axes, starting with the animal-vegetal axis that will be divided into the three germ layers endo-, meso- and ectoderm from vegetal to animal. In a temporally overlapping process, these three germ layers then become patterned along their dorso-ventral and later antero-posterior and left-right axes. Although the precise molecular control mechanisms that underlie the patterning are not understood yet, a number of protein interactions have been discovered that play essential roles in the establishment of embryonic axes and later in the formation of the organism (for review see Lebreton et al., 2006).

During cortical rotation after sperm entry, RNA encoding Wnt11 is shifted from the vegetal pole towards the future dorsal side, where its protein diffuses into the dorsal egg cytoplasm (Figure 1) (Heasman et al., 2001; Ku and Melton, 1993). Together with the HSPG and FRL1 cofactors, Wnt11 was suggested to activate the canonical Wnt pathway that includes downstream effectors like dishevelled, β-catenin and GSK3 as well as the transcription factor siamois (Figure 3) (Jessen and Solnica-Krezel, 2005; Tao et al., 2005). Dishevelled is an inhibitor of GSK3, which initially is distributed equally throughout the egg. Since GSK3 itself represses the expression of β-catenin, β-catenin will only be active on the dorsal side where it stays stable (Heasman, 2006; Kloc and Etkin, 1995). β-catenin works in a complex with another transcription-factor, Tcf3, to promote activation of target genes like siamois that is essential for dorsal axis formation (Laurent et al., 1997). If the β-catenin binding site for interaction with Tcf3 is mutated, embryos do not develop a dorsal axis (Fagotto et al., 1997; Molenaar et al., 1996). At the start of zygotic transcription, some hours before gastrulation, a direct target gene of the canonical Wnt-signalling pathway, siamois, shows maximal expression at the Nieuwkoop centre, from where siamois is capable of inducing the Spemann organizer that develops in direct proximity animal to it (Moon and Kimelman, 1998). Named after the Dutch embryologist, the Nieuwkoop centre describes a dorso-vegetal area of the blastula that is capable of inducing dorsal mesodermal tissues in the overlying animal cells, the first step towards dorso-ventral patterning of mesoderm (Gerhart et al., 1981; Nieuwkoop, 1973). The Wnt signalling effector, siamois, binds to the promoters of, and activates, organizer-genes such as gsc, Xlim-1, frzb and cerberus, the last of which is an inhibitor of BMP and nodal signals. It activates these genes in combination with transcription factors of

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the TGFβ signalling pathway, which is activated by Vg1 or Xenopus nodal related (Xnr) proteins (Moon and Kimelman, 1998; Nishita et al., 2000).

Figure 3: Canonical Wnt signalling pathway. Wnt protein binds to its receptor, a member of the Frizzled family

of proteins. Frizzled then activates Dishevelled, allowing it to become an inhibitor of glycogen synthase kinase 3 (GSK3). Active GSK3 would prevent the dissociation of β-catenin from the APC protein. So by inhibiting GSK3, the Wnt signal liberates β-catenin to associate with a LEF or TCF protein and become an active transcription factor.

The maternal RNAs of Vg1 and VegT have been shown to localize tightly to the vegetal cortex in the oocyte (Weeks and Melton, 1987; Zhang and King, 1996). Both proteins are essential for mesoderm induction (Agius et al., 2000; Harland and Gerhart, 1997; Heasman et al., 2001) and are thought to diffuse from the site of anchored RNA. The TGFβ ligand Vg1 later becomes secreted from the vegetal cells in early development and functions in a concentration dependent manner to activate organizer genes including BMP antagonists (Birsoy et al., 2006; Harland and Gerhart, 1997; Xanthos et al., 2001). A gradual distribution of these proteins enables the induction of a threshold response along this gradient, which gives a positional identity to each cell in the field of influence. Proteins that pattern embryonic morphology in this way are called morphogens and will be described in detail below. The diffusion of the transcription factor VegT is limited to the vegetal half of the embryo by the third cell division that divides the embryo into animal and vegetal halves. VegT can induce Xnrs 1, 2, 4, 5 and 6, which are relevant for mesoderm induction as well as derriére, another member of the TGFβ superfamily (Kofron et al., 1999; Takahashi et al.,

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2000). The overlap of the high points of the Wnt and VegT gradients marks the Nieuwkoop centre at the dorso-vegetal side (Figure 5).

The organizer was first described by Hilde Mangold and Hans Spemann in 1924. They showed that dorsal mesoderm from an early gastrula, when grafted to the ventral side of another embryo, induces a secondary embryonic axis in the host embryo. The organizer acts as a signaling center for both antero-posterior and dorso-ventral patterning and has the ability to dorsalize mesoderm (Spemann, 1924) (Agius et al., 2000). Equivalents of the Spemann’s organizer have been identified in many vertebrate species; these equivalents include the node in mouse and chicken, and the embryonic shield in fish (Figure 4).

Figure 4: The inductive properties of the Spemann organizer. A graft of the organizer region, from the dorsal lip

of the blastopore of an early Xenopus gastrula to the ventral side of another early gastrula embryo, results in the development of an additional anterior axis at the site of graft. (After Wolpert L., 2007)

Just vegetal to this organizer tissue in the Xenopus embryo the formation of bottle cells indicates the start of gastrulation movements on the future dorso-posterior side of the embryo. These cell shape changes are induced by a variety of growth factors that all belong to the TGFβ protein family including Xnr1, 2, 4, 5, 6 and Derrière. Being synthesised and secreted from the dorsal organizer, TGFβ proteins were suggested to form gradients towards the ventral side of the gastrula and to induce mesoderm formation and patterning along this gradient (Figure 5). Regions with minimal nodal signalling become ventral mesoderm, while medium Xnr function turns animal tissue into dorsal mesoderm, and the region of highest Xnr expression defines the organizer (Agius et al., 2000). These factors are called morphogens, owing their special ability to spread over tissue and to induce a variety of mesodermal tissues at a distance depending on their concentration.

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Figure 5: Animal-vegetal and dorso-ventral patterning of the Xenopus blastula. Vegetally secreted proteins such

as VegT and Vg1 induce the animal tissue in the margin to become mesoderm. Dorsal morphogens such as Wnt11 in the Nieuwkoop centre induces the organizer animal to it. Organizer proteins such as Xnrs and Derrière pattern mesoderm and endoderm in the dorso-ventral axis.

1.2 Morphogens

The principle of morphogen function is of great interest for developmental biology and medicine because it is an essential process, initiated mostly by one type of protein, that if interrupted during embryogenesis leads to severe phenotypes that are always lethal. It comes as no surprise that the process as a whole is complex and has been subject to many studies for decades.

1.2.1 History

Seventy years ago, Pasteels and Dalcq first spoke about signals that spread from “organizing tissues” and induce distinct cellular responses. The question about how this positional information is established, however, is still a matter of intense discussion and research (Affolter and Basler, 2007; Ashe and Briscoe, 2006; Kerszberg and Wolpert, 2007; Lander, 2007; Pasteels, 1937; Smith et al., 2008; Strigini, 2005). Alan Turing coined the name “morphogen” when he proposed his reaction-diffusion model that describes how the interaction between an agonist and an antagonist determines the size and fate of a field of cells. This model can explain for example the fur pattern of the zebra (Turing, 1952) (see also: http://www.psych.utah.edu/stat/dynamic_systems/Content/docs/e42/02-09-21_N100-K2-SR_zebra-L1-5in.html).

Nieuwkoop, a pioneer in Xenopus embryology that did describe the Nieuwkoop centre, mentioned above, also performed an important series of experiments where he showed that mesoderm differentiation (prospective notochord, muscle, and blood) could be induced by non-cell autonomous signals (Nieuwkoop, 1973). He made “sandwiches” of vegetal pole explants (prospective endoderm) and animal caps (prospective ectoderm). Animal caps alone

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develop into epidermis and express all the molecular as well as morphologic features of skin. Before knowing any molecular details, Nieuwkoop showed that the vegetal poles induced the animal caps to become mesoderm instead of skin (Nieuwkoop, 1967a; Nieuwkoop, 1967b). One explanation for this result was the existence of a secreted factor produced by vegetal cells that spreads into the animal hemisphere providing positional information to the more marginal cells. These cells respond by differentiating into mesoderm while animal pole cells at a greater distance from the factor’s source develop according to their default fate into ectoderm.

1.2.2 Definition

In 1969, Lewis Wolpert described many aspects of morphogen actions in his “French flag model” (Wolpert, 1969). According to him, a “morphogen” by definition must fulfil two key criteria: it must act directly at a distance, and it must induce distinct outputs at different concentrations. In theory, all it takes for the specification of different fates across a field of cells is a defined location for the source of a soluble substance, the morphogen, from which it can spread over a distance. Constantly produced on one end and absorbed at a “sink” on the other, it will form a gradient. In practice, the production site could be represented by a defined area of morphogen-secreting cells like the vegetal cells in the Xenopus blastula. The concept of a “sink” would be harder to imagine if we presume that no protein is lost through the outer layer of epithelial-like cells in a Xenopus blastula (Chalmers et al., 2003; Johnson and Ziomek, 1981) (reviewed by (Johnson and McConnell, 2004); (Muller and Bossinger, 2003)). Nevertheless, the same gradual effect can also be achieved by a constant rate of ligand degradation along its path of action. Being sensitive to the morphogen’s concentration, the responding cells will react in different manners according to the concentration they perceive. This is expressed in Wolpert’s “French flag model” in the way that cells respond with different colours at different distances from the morphogen source. Cells closest to the source will produce blue colour, because they experience a high concentration level of the morphogen; cells further away will transform their positional information into white and cells furthest away receiving the lowest concentration levels of the signalling molecule respond as red cells (Wolpert, 1969; Wolpert, 1971) (Figure 6)

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Figure 6: “French flag model” of morphogen action. Secreted morphogen (green dots) is released (arrow) from a

restricted region (green cells) and forms a spatial gradient of concentration. Different target genes are expressed at different concentration thresholds (c1, c2 and c3). At c1, target-gene x (blue) is activated; at c2, target-gene y (white) is activated and at c3 target-gene z (red) is activated. The pattern resembles the colours of the French flag (Adapted from Entchev E.V. & Gonzalez-Gaitan M.A., 2002).

1.2.3 Concept

In practise, the whole process of morphogen signalling involves several (probably independent) steps. First, the morphogen has to be processed and secreted properly; second, it has to travel to its final destination while establishing a concentration gradient; and finally, and importantly, the ligand has to activate the appropriate signalling pathway in the right cells. Along this signalling pathway, the decision has to be made about the correct target-gene response for the number of morphogen molecules that have reached this particular cell. One would expect that the morphogen is produced by a defined region of cells in which the time and rate of secretion is coordinated appropriately. Via active or passive transport, the ligand would spread across the surrounding tissue while a stable gradient could be established by a constant rate of degradation during progression. One way in which cells might respond differently to different concentrations of morphogens does involve differential sensitivities of target gene promoters to different morphogen cues. For example, the promoter of hunchback,

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a target gene of the morphogen Bicoid that patterns the Drosophila embryo in early syncytial stages responds to direct cooperative binding of multiple Bicoid molecules (Driever et al., 1989). The morphogen Bicoid is an exception because it functions directly as a transcription factor. That is only possible in the particular case of early Drosophila development where the embryo consists of a syncytium.

1.2.4 Unanswered questions

At present, the most intensely studied process in morphogen signalling is the passage of the ligand itself. A variety of models have been proposed for the different morphogens in various developmental contexts. For example, the movement of decapentaplegic (dpp), a TGFβ protein, in the imaginal wing disc epithelium of Drosophila embryos has been suggested to pass through neighbouring cells by transcytosis or via cell extensions. In the same type of tissue, wg and hh family members appear to depend on a variety of glycoproteins for their proper transport through the extracellular space. In mesenchymal tissue of the frog blastula, simple diffusion has been suggested for the spreading of Activin. A well studied exception from these examples of morphogens that function in multicellular tissues is the passive diffusion of Bicoid in the syncytium of an early Drosophila embryo. Some of these models will be discussed in more detail in the chapters “Morphogen examples” and “Morphogen progression and gradient establishment”.

The way morphogens travel is important for the understanding of the individual establishment of a gradient and its interpretation by responding tissues. However, none of these models has yet explained the gradual distribution of a morphogen to complete satisfaction. In part, this is because the process of gradient establishment has to fulfil several criteria like robustness and flexibility at the same time. Robustness describes the ability of a morphogen gradient to establish itself despite perturbations and to coordinate long-range patterning. This means that the variability of a gradient slope has to be minimal in order to stay in the range of threshold interpretation. This question has been examined for the case of the distribution of dpp and its target gene spalt in the Drosophila wing disc epithelium, where the study showed sufficient stability in ligand distribution (Bollenbach et al., 2008). Flexibility, on the other hand, is a consequence of the separation between positional information (the morphogen gradient itself) and the interpretation of that information by cells. The same morphogen at the same concentration can cause a very different response in different environments. For example, tissues can use the same molecular coordinate system dpp and BMP to determine their

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dorso-ventral position and fate in flies and vertebrates respectively, even though the structures that emerge are very different (Ben-Zvi et al., 2008; Green, 2002; Lawrence et al., 2007).

1.3 Morphogen examples

Over the years, a large variety of morphogens have been identified and their functions thoroughly investigated. Signal gradients perform their different functions in different types of tissue by activating a subset of genes to create molecular and cellular patterns. One vivid example of this patterning is eyespot formation in the butterfly wing with up to five different ring-colours (Brakefield, 2003). The molecules involved in the formation of these circular structures are still being discovered. However, the few that have been identified so far are known targets of hedgehog (hh) and wingless (wg, a Wnt-1 homologue), two morphogens with functions previously described in vertebrate and insect development (Estella et al., 2008; Monteiro et al., 2007). Interestingly the butterfly eyespot is reminiscent of experimental results in frog and fish embryos where local morphogen expression in an exogenous place leads to the ring-like expression of various target genes (Papin and Smith, 2000; Williams et al., 2004) (Gurdon et al., 1994) (Figure 7).

Figure 7: Eyespots of the butterfly wing as example for morphogen function. (A) Wing pattern of a

gynandromorph individual of the butterfly Bicyclus anynana. The left side is male, and the right side, female (Taken from Brakefield, 2003). (B) Clonal injection into one cell of an early blastula Xenopus embryo. (C) Red patch of cells overexpressing Xnr2 induce exogenous expression of Xbra as blue ring of in situ hybridized transcript.

In contrast to whole tissue field patterning it has also been proposed that gradients control the orientation of cells within a field (Lawrence, 1966; Stumpf, 1966). The way in which morphogen gradients provide both scalar and vectorial information to cells is possibly even definitive for true morphogens (Lawrence, 2001; Wolpert, 1969). Although a latent polarity can sometimes be revealed by experiments, some cell types openly display their polarity by the orientation of hairs or cilia, a property called planar cell polarity (PCP). Recent progress has been made in understanding the detailed molecular mechanisms of planar polarity in

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Drosophila cuticles (See (Lawrence et al., 2007) for review). The emerging model proposes that graded extracellular signals provide a bias for the movement of specific molecules towards one or the other end of a cell and that these intracellular distributions are reinforced by very local positive and negative feedback loops.

There are a great number of examples where morphogens have an important function in development. These include patterning both of whole embryos and of single organs. In this chapter, only some of the most studied molecules with morphogen character will be highlighted.

1.3.1 Hedgehog (Hh)

More than 30 years ago, elegant microsurgery experiments suggested that a group of posterior mesenchymal cells in the developing vertebrate limb (on the little finger side) known as the zone of polarizing activity (ZPA) is able to control the pattern of digits (Maccabe et al., 1973; Smith, 1979; Smith et al., 1978; Tickle et al., 1975). Retinoic acid was an early candidate for this positional information. Eventually it became clear that the activity of retinoic acid is mediated by secondary signals and it is now known that it induces the expression of sonic hedgehog (shh), an orthologue of hh in Drosophila (Riddle et al., 1993). Shh signalling is dose-dependent, inducing different digits at different concentrations and it produces long-range effects in the limb. There is however no formal proof yet that shh acts directly on cells or via a secondary signal (Figure 8). Some evidence points to an involvement of BMP and FGF in this process (Khokha et al., 2003; Robert, 2007; Zuniga et al., 1999).

However, in the vertebrate neural tube, experiments have demonstrated that Shh acts directly to pattern the dorso-ventral axis. A ventral source of Shh signal in the midline organizes different neural subtypes at distinct dorsal-ventral positions (Ericson et al., 1997). Ectopic expression of a mutated form of the Shh receptor, Patched (Ptc), which does not bind Shh but does antagonize its signaling, causes cell-autonomous ventral-to-dorsal switches in neural progenitor identity (Briscoe et al., 2001), strongly suggesting that Shh functions by acting on cells directly (Figure 8).

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Figure 8: Hh/shh morphogen function in different developmental environments. (A) Ectopic production of Shh

in the chicken wing bud, induced by implanting shh expressing cells into the anterior limb bud, induces a mirror image duplication of the wing structure. Shh is expressed in the region corresponding to the ZPA in the wing bud (top). Implanted cells that ectopically produce Shh in the anterior of the limb bud induce a mirror image duplication of the wing structure (bottom). Digits (II, III and IV) are labeled on the schematic, and radius (R), ulna (U) and humerus (H) are labeled in the photographs on the right. (B) Ectopic expression of hh in the imaginal wing disc of Drosophila . By making a clone of cells expressing hh, the protein induces a mirror image duplication of the anterior wing structure. Hh produced in the P compartment is secreted into the A compartment (top). A clone of cells ectopically expressing hh in the A compartment induces a complete mirror image duplication of the A compartment (bottom). (C) A model for effects of Patched (Ptc) on neural patterning. (I; left half) Shh emanating from the notochord (N) induces formation of the floor plate (FP), and subsequent shh expression in the FP generates a ventral-dorsal activity gradient of Shh (as indicated by the density of the blue dots). (II; left half) The activity gradient of Shh promotes the specification of a series of ventral cell types: p0, p1, p2, pMN and p3, which are progenitor domains from which distinct V0 neurons, V1 neurons, V2 neurons, motoneurons and V3 neurons are generated respectively. Production of a mutated form of the Shh receptor Ptc (Ptc1Dloop2; I; right half; light green), which does not bind Shh but antagonizes its signaling, causes cell-autonomous abnormal dorsal spread of Shh and (II; right half) ventral-todorsal switches in neural progenitor identity. (After Tabata & Takei, 2004).

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1.3.2 Bone morphogenetic protein (Bmp )

Another function of hedgehog is in the antero-posterior patterning of the Drosophila wing disc epithelium (Figure 8/9). One of the targets of hh in the imaginal wing disc of the fruit fly is dpp, a BMP homologue. Dpp functions here as a second morphogen that patterns the wing beyond its the central domain of expression in the antero-posterior axis (Lecuit et al., 1996; Nellen et al., 1996). The disc is divided into anterior and posterior compartments: the diffusible protein hh is expressed in the posterior compartment. Dpp is expressed at the compartment boundary and induces several target genes including sal and omb, with omb being expressed in a wider domain than sal (Figure 9).

Figure 9: Hh, dpp and wg morphogens pattern the Drosophila wing. (A) Distribution of Hh, Dpp-GFP and wg in

the wing imaginal disc. Note the graded distribution away from the expressing domain. (B) Hh produced in the posterior (P) compartment generates a short range gradient of Hh in the anterior (A) compartment. Hh both patterns the central domain of the wing and induces the expression of en, ptc and dpp, at high, middle and low thresholds, respectively, in a stripe of cells adjacent to the AP compartment boundary. Note that en is induced by Hh in the anterior compartment in late larval development. Dpp induces expression of sal and omb at high and low thresholds, respectively, and patterns the wing beyond the central domain (After Tabata & Takei, 2004).

Evidence that dpp acts directly on cells in a concentration dependent manner, rather than by acting through a signal relay mechanism, comes from experiments that used a constitutively active form of the dpp receptor (Lecuit et al., 1996; Nellen et al., 1996). Dpp binds to a type II serine/threonine kinase receptor which together with a type I receptor (thickveins, tkv),

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activates the receptor regulated signal transducer Mad. Mad translocates to the nucleus upon phosphorylation by the type I receptor to regulate the expression of target genes (e.g. sal and omb). A constitutively active form of tkv (tkv*), when ectopically expressed, can induce the expression of the target genes sal and omb. The fact that dpp functions as a morphogen was shown by the strictly cell-autonomous effects of tkv* in inducing expression of sal and omb. If dpp were to trigger a signaling relay mechanism, the effects of overexpressing tkv* should be non-autonomous because a second signal emanating from the cells overexpressing tkv* would also affect surrounding cells. In addition, different levels of dpp upregulate the different targets, Omb and Sal (Lecuit et al., 1996; Nellen et al., 1996).

1.3.3 Fibrobla st grow th factor (Fgf )

The morphogen function of embryonic fibroblast growth factor (eFgf ) in mesoderm induction was identified in Xenopus at about the same time as the potential of Activin to pattern and induce mesoderm long-range (Slack et al., 1987) (Kimelman and Kirschner, 1987). EFgf has been suggested to induce ventro-posterior fates by acting in an animal-to-vegetal gradient (Green et al., 1992). Dose–response experiments indicated that Fgf might be a morphogen because different doses induced different cell types (Green et al., 1990; Green and Smith, 1990; Slack et al., 1987). At gastrulation stages eFgf provides a positive feedback loop for the nodal-induced mesodermal gene Xbra (Cornell and Kimelman, 1994; Cornell et al., 1995; LaBonne and Whitman, 1994; Schulte-Merker and Smith, 1995) (Isaacs et al., 1994).

Fgf has also been proposed to control activation of the segmentation process in the presomitic mesoderm (PSM) of vertebrate embryos. In chicken and mouse embryos, Fgf8 has been suggested to act as a morphogen in an unconventional fashion through building a gradient of progressive RNA decay in the growing posterior tip of the embryo (Dubrulle and Pourquie, 2004a; Dubrulle and Pourquie, 2004b). This Fgf8 mRNA gradient is translated into a gradient of Fgf8 protein, which correlates with graded phosphorylation of the kinase Akt, a downstream effecter of Fgf signalling. Such a mechanism provides an efficient means to monitor the timing of Fgf8 signalling, coupling the differentiation of embryonic tissues to the posterior elongation of the embryo. (reviewed in (Smith and Gurdon, 2004))

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1.3.4 Activin & nodals

This thesis will focus on the family of TGFβ molecules as an example of morphogen function in early Xenopus laevis development, in particular during mesoderm induction and patterning. In this thesis, we used Activin as a representative of mesoderm inducing TGFβ molecules, to address questions about morphogen progression and signalling.

In the early 1970s, the group of H. Tiedemann isolated a mesoderm-inducing factor from chicken embryonic tissue that was able to transform the prospective ectoderm of the newt Triturus into mesoderm (Kocher-Becker and Tiedemann, 1971) (Geithe et al., 1975). J. C. Smith was then able to extract a similar mesoderm inducing factor (MIF) from a Xenopus tadpole cell line that was not only able to induce the differentiation of animal tissue into mesoderm, but also to do this in a concentration dependent fashion (Smith, 1987). MIF turned out to be Activin, a member of the transforming growth factor family (Asashima et al., 1990; Smith et al., 1990) (Albano et al., 1990). In succession, a series of other growth factors were identified as mesoderm inducers, including Fgf and the transforming growth factor Vg1. While a gradual Fgf function mimicked the ventroposterior fate pattern of Xenopus mesoderm, Activin could induce dorsal mesoderm (Green and Smith, 1990; Symes, 1987). Strikingly, Activin was not only able to induce dorsal mesodermal tissue types but also characteristic cell movements and elongation of explants reflecting the convergent extension of endogenous dorsal mesoderm (Symes, 1987). Furthermore, as concentrations of Activin increased, it proved to be able to induce three different tissue types representing positions along the antero-posterior embryonic axis: Presumptive ectodermal tissue from the animal cap of a Xenopus laevis blastula was transformed into epidermal and mesenchymal tissue after induction with a low concentration of Activin protein. The same tissue differentiated into muscle and neural tissues with a higher concentration and underwent transformation into notochord, cement gland and neural development when induced with an even higher concentration of Activin (Green et al., 1990; Green and Smith, 1990; Smith et al., 1988). The low-to-high sequence of induced markers corresponded to the posterolateral-to-dorsoanterior sequence of tissues observed in cell fate experiments of the Xenopus blastula (Green et al., 1992). These results appeared to resemble the gradual response to a secreted mesoderm patterning factor. So far, at least eight secreted members of the TGFβ family with the ability to induce mesoderm and axial patterning in Xenopus have been described. These include Vg1 as well as Xnr1, 2, 4, 5, 6, Activin and derriére that are each to some extend essential for the proper formation of the body axis (Birsoy et al., 2006; Jones et al., 1995; Joseph and Melton, 1997; Piepenburg et al., 2004; Smith, 1995; Sun et al., 1999; Takahashi et al., 2000; White et al., 2002) (Figure 10).

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Figure 10: Xenopus late blastula fate map. The ectoderm gives rise to the epidermis and nervous system. Along

the dorso-ventral axis the mesoderm gives rise to head mesoderm, notochord, somites, heart, kidney, and blood. In

Xenopus, there is also endoderm overlying the mesoderm in the marginal zone (compare Figure 2).

The nodal genes are highly conserved throughout the vertebrates. Studies in other vertebrate models have shown that the nodal signalling pathway, including nodal homologues and downstream signaling components, is essential for embryonic development. Mutation of the single copy of nodal in the mouse causes a loss of embryonic mesoderm (Brennan et al., 2001).Squint, one of two nodal homologues in zebrafish, has the ability to signal long-range for mesoderm induction and patterning by regulating different target genes in a concentration-dependent manner (Chen and Schier, 2001). The direct action of squint was demonstrated by experiments taking advantage of mutations in an essential nodal coreceptor, one eyed pinhead (oep). A single cell injection of squint RNA into a oep mutant blastula embryo caused target gene induction in wild-type cells implanted distantly from the squint-injected cell. Thus, although the mutant cells cannot transduce the squint signal, wild-type cells placed at a distance from the source of squint still respond to the nodal homologue (Chen and Schier, 2001).

Despite the discovery of the mesoderm inducing activities of Activin in Xenopus (Asashima et al., 1990; Smith et al., 1988); see above) deciphering its in vivo role has been difficult. The murine knockout of both activin subunits, Activin βA and βB, resulted in viable embryos that only died shortly after birth without any obvious gastrulation phenotype (Vassalli et al., 1994). In frog embryos, the resulting phenotype from experiments using a truncated Activin type II receptor suggested that Activin signalling is essential for mesoderm development not taking in account that the activity of other TGFβ proteins is also dependent of this particular receptor (Dyson and Gurdon, 1997). In 2000, Agius et al. could show that, by interfering only with Xnrs, signalling in Xenopus leads to complete mesoderm ablation suggesting that Xnrs might be the only essential proteins for mesoderm formation in frog development (Agius et al., 2000; Dyson and Gurdon, 1997). It is, however, worth noting that the ablation of the

Long-range signalling of TGF\03B2 morphogens in the early Xenopus embryo

Universidade de Lisboa

Faculdade de Ciências de Lisboa

Departamento de Biologia Animal

Long-range signalling of TGFβ morphogens in the

early Xenopus embryo

Anja Irene Hagemann

Doutoramento em Biologia

(Biologia do Desenvolvimento)

Universidade de Lisboa

Faculdade de Ciências de Lisboa

Departamento de Biologia Animal

Long-range signalling of TGFβ morphogens

in the early

Xenopus

embryo

Anja Irene Hagemann

Tese orientada por:

Professora Doutora Sólveig Thorsteinsdóttir

e Professor Doutor Jim C. Smith

Doutoramento em Biologia

(Biologia do Desenvolvimento)

Os trabalhos apresentados nesta tese foram realizados com o apoio financeiro da Fundação para a

Ciência e a Tecnologia (bolsa de referência SRFH/BD/11807/2003) e, posteriormente, da Fundação

NOTAS PRÉVIAS

CONTENTS

I

R

D

ACKNOWLEDGEMENTS

RESUMO

ABSTRACT

LIST OF ABBREVIATIONS

1.1

Early Xenopus development

1.1.1

Morphological pola risation in earl y development

of the

Xenopus

embryo

1.1.2

Molecular contro l of axis fo rmatio n of the

Xenopus

embryo

1.2

Morphogens

1.2.1

History

1.2.2

Definition

1.2.3

Concept

1.2.4

Unanswered questions

1.3

Morphogen examples

1.3.1

Hedgehog (Hh)

1.3.2

Bone morphogenetic protein (Bmp )

1.3.3

Fibrobla st grow th factor (Fgf )

1.3.4

Activin & nodals