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PKD2-dependent sensing mechanism in Kupffer’s vesicle: how it affects the left-right axis establishment in zebrafish
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RESUMO: Estabelecimento do eixo Esquerda-Direita (ED) é um processo complexo que ocorre cedo
durante o desenvolvimento. Exige a integração de várias vias de sinalização, tais como TGF-
β, Notch, Wnt e Cálcio. Também envolve a coordenação de dinâmica de fluídos, difusão de
morfogénios e movimento ciliar. Tudo junto e coordenado no tempo leva à correta localização
dos órgãos internos. Problemas no estabelecimento deste eixo estão normalmente
associados a doenças crónicas. As primeiras decisões assimétricas acontecem numa
pequena e transiente estrutura chamada o Organizador Esquerda Direita, uma estrutura ciliar
que existe em vários vertebrados. Cílios móveis geram um fluxo de fluido assimétrico que se
traduz numa expressão génica assimétrica entre o lado esquerdo e o lado direito. Estes sinais
são depois transferidos à Mesoderme Lateral, o tecido que mais tarde dá origem ao coração
e influencia a endoderme que dá origem a órgãos como o fígado e o pâncreas.
No Capítulo 2, focámo-nos em tentar compreender como é que as vias de sinalização se
coordenavam para decidir entre um cílio móvel e imóvel no Organizador. Embora a expressão
de Foxj1a em todas as células do Organizador produza cílios com ultra-estrutura compatível
com motilidade, a decisão entre mover ou não é exclusiva da via de sinalização Notch. A
seguir, focámo-nos em caracterizar Pkd2, um canal de cálcio importante no estabelecimento
do eixo ED. Este canal está associado ao Pkd1l1, uma molécula com domínios capazes de
sentir fluido e responder com entrada de cálcio na célula. No Capítulo 3, questionámos se não
ter fluxo no Organizador tinha o mesmo impacto que não ter um mecanismo para sentir esse
fluxo através da ausência do Pkd2. A única manipulação que não afetou a arquitetura do
Organizador foi remover o Pkd2 apenas nas células do Organizador, o que não é muito
eficiente e tem impacto na velocidade do fluxo dentro do Organizador. Ainda assim, todas as
manipulações efetuadas deram o mesmo fenótipo: randomização da posição dos órgãos. No
Capítulo 4, fizemos um estudo de transcritómica usando apenas as células do Organizador em embriões normais ou injetados com morpholino contra o Pkd2. Embora não tenhamos
encontrado genes com expressão assimétrica à volta do Organizador, encontramos quatro
novos genes que influenciam o estabelecimento do eixo: cacybp, frzb, pvalb6 e ncl1. No
Capítulo 5, focámo-nos na ncl1, um gene nunca antes associado ao estabelecimento do eixo
e que atua como antagonista da via de sinalização TGF-β ao influenciar a secreção de Lefty
e influenciando a padronização da mesoderme-endoderme. Nós descobrimos que este gene
impactua no estabelecimento do eixo, provavelmente ao influenciar a secreção de algum
elemento da via de sinalização TGF-β. No Capítulo 6, focámo-nos em estabelecer o peixezebra
como um bom modelo para estudar toxicidade no rim em resposta a fármacos.
Em suma, os resultados apresentados nesta tese providenciam novas pistas para o
estabelecimento do eixo ED, desde o movimento do cílio a novos genes a jusante do Pkd2 e
do cálcio. Também reforçámos a ideia de que o peixe-zebra pode ser um bom modelo para
estudar doenças humanas.
ABSTRACT: Left-Right (LR) axis establishment is a complex process that happens early in development. It requires the interplay of several genetic pathways like TGF-β, Notch, Wnt and Calcium signalling. It also involves the integration of fluid dynamics, morphogen diffusion and cilium biosynthesis to correctly position the internal organs in their final destinations. Problems in LR axis establishment are often associated with chronic diseases. The first asymmetric decision commonly happens in a small transient structure, the Left-Right Organizer (LRO), a ciliated structure present in many vertebrates. Motile cilia generate an asymmetric fluid flow that is perceived differently between the left and the right side, which generates a calcium response and asymmetric gene expression. These signals are then transferred to the Lateral Plate Mesoderm, the tissue that will later give rise to the heart and influence the endoderm derived organs such as the liver and pancreas. In Chapter 2, we focused in understand the pathways behind deciding between being a motile vs immotile cilium in the LRO. Although all cilia are made motile in terms of ultrastructure due to Foxj1a expression, the decision to move or not is dependent on Notch signalling alone. Then, we focused on further characterization of an important calcium channel, Pkd2, in the LR. This channel is thought to partner with Pkd1l1 and sense flow, an important feature in LR. In Chapter 3, we asked if having no flow had the same impact as having no Pkd2-mediated sensing. The only manipulation that did not affect the LRO architecture was to target Pkd2 on the LRO cells only, which still left visible Pkd2 protein and a slower flow. Still, all our manipulations had the same phenotype: randomization of organ situs. In Chapter 4, we performed a LRO-specific microarray between WT and pkd2 morphant embryos in order to find other asymmetric genes present in the LRO. Although we did not find any gene expressed asymmetrically between left and right side, we indeed find four new genes with minor roles in LR: cacybp, frzb, pvalb6 and ncl1. In Chapter 5, we further focused in manipulating ncl1, a new gene in LR that is known to act as a TGF-β antagonist by facilitating Lefty secretion and impacting on mesendoderm patterning. Indeed, we found that it has an impact on LR, probably by influencing the secretion of some TGF- β signalling player. In Chapter 6, we set to establish zebrafish as a good model to study kidney toxicity when metabolizing drugs. Together, the results presented in this thesis provide new clues for LR axis establishment, from cilia motility to new downstream genes of Pkd2 and calcium. It also highlights the zebrafish as a good model to study human disease.
ABSTRACT: Left-Right (LR) axis establishment is a complex process that happens early in development. It requires the interplay of several genetic pathways like TGF-β, Notch, Wnt and Calcium signalling. It also involves the integration of fluid dynamics, morphogen diffusion and cilium biosynthesis to correctly position the internal organs in their final destinations. Problems in LR axis establishment are often associated with chronic diseases. The first asymmetric decision commonly happens in a small transient structure, the Left-Right Organizer (LRO), a ciliated structure present in many vertebrates. Motile cilia generate an asymmetric fluid flow that is perceived differently between the left and the right side, which generates a calcium response and asymmetric gene expression. These signals are then transferred to the Lateral Plate Mesoderm, the tissue that will later give rise to the heart and influence the endoderm derived organs such as the liver and pancreas. In Chapter 2, we focused in understand the pathways behind deciding between being a motile vs immotile cilium in the LRO. Although all cilia are made motile in terms of ultrastructure due to Foxj1a expression, the decision to move or not is dependent on Notch signalling alone. Then, we focused on further characterization of an important calcium channel, Pkd2, in the LR. This channel is thought to partner with Pkd1l1 and sense flow, an important feature in LR. In Chapter 3, we asked if having no flow had the same impact as having no Pkd2-mediated sensing. The only manipulation that did not affect the LRO architecture was to target Pkd2 on the LRO cells only, which still left visible Pkd2 protein and a slower flow. Still, all our manipulations had the same phenotype: randomization of organ situs. In Chapter 4, we performed a LRO-specific microarray between WT and pkd2 morphant embryos in order to find other asymmetric genes present in the LRO. Although we did not find any gene expressed asymmetrically between left and right side, we indeed find four new genes with minor roles in LR: cacybp, frzb, pvalb6 and ncl1. In Chapter 5, we further focused in manipulating ncl1, a new gene in LR that is known to act as a TGF-β antagonist by facilitating Lefty secretion and impacting on mesendoderm patterning. Indeed, we found that it has an impact on LR, probably by influencing the secretion of some TGF- β signalling player. In Chapter 6, we set to establish zebrafish as a good model to study kidney toxicity when metabolizing drugs. Together, the results presented in this thesis provide new clues for LR axis establishment, from cilia motility to new downstream genes of Pkd2 and calcium. It also highlights the zebrafish as a good model to study human disease.
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Estabelecimento do eixo Esquerda-Direita Pkd2 Motilidade ciliar Via de sinalização TGF-β e Notch Left-Right axis establishment Pkd2 Cilia motility TGF- β signalling, Notch signalling