Testing the role of Extracellular Vesicles in early Left-Right Patterning

Financiador

Organização

Publicações

Testing the role of extracellular vesicles in early left right patterning

Publication . Pestana, Sara; Lopes, Susana; Barral, Duarte C.

Abstract Bilaterian animals, such as humans, are characterized by an external roughly mirror symmetry along the left – right axis that covers a pronounced internal asymmetric arrangement of the thoracic and abdominal organs. While external symmetry has been associated with health and beauty standards, the internal asymmetry may rely more on efficiency and functionality of the different physiological systems. The left – right asymmetry of visceral organs is established early on during embryonic development within a transient and specialized structure, commonly referred to as the left – right organizer (LRO). The LROs appear in many shapes and sizes, depending on the species, but a common feature in some vertebrates is the requirement of motile cilia. The movement of these tiny hair-like protrusions generate a directional fluid flow, that scales with the cube of cilia length, in order to become capable of triggering a differentiated response on the left side of the LRO. Such flow-dependent response involves Pkd2 channel activation and calcium signaling that subsequently drive the left sided expression of the Nodal signaling cascade. Nodal is a secreted protein that translates the asymmetries established at the LRO to the rest of the embryo, through the lateral plate mesoderm. As embryonic development evolves, at specific time points and locations along the anterior – posterior axis, Nodal induces the expression of genes involved in the formation of the heart, brain, gut and its derivatives, modulating the lateralization of these organs. With this work, we dedicated our efforts to understanding a few molecular and cellular steps missing in the establishment of the left – right axis within the LRO. In the Chapter 2, we explored how the fluid flow is sensed by the LRO cells. Between the two hypotheses in the field, one based on mechanosensing and other on chemosensing properties of the flow, we found that the number of extracellular vesicles is too low and variable to transport sufficient and efficiently a sidedness molecular signal towards the left sided LRO cells. Moreover, pharmacological impairment of distinct endocytic pathways did not impact on heart laterality arrangement. We also found out an upstream regulator of Notch signaling, syntenin-a, involved in the cell fate decision between motile and immotile cilia. We showed that syntenin-a loss-of-function severely affected the left – right axis development. By downregulating the levels of syntenin a, Notch signaling is activated increasing the expression of her12 and resulting in a higher number of immotile cilia, in concordance with our previous published data. We next described a potential molecular switch, downstream of Notch signaling, composed by the Rabconnectin complex. As this complex is known to promote V-ATPase assembly and consequently its activity, we inhibited the V-ATPase activity and we observed an increase in the number of motile cilia. Thus, suggesting that the link between Notch signaling and motile – immotile cilia ratio is through the modulation of pH. Lastly, in Chapter 3, we focused on the impact of ciliary dysfunction in the epithelial respiratory cells. We characterized the distribution pattern of several ciliary proteins in two siblings harboring a primary ciliary dyskinesia causing mutation on Zmynd10 gene. Recent studies showed that ZMYND10 is one of the cytoplasmatic factors responsible for stabilizing and driving axonemal dynein arm assembly. We showed here that outer and inner axonemal dyneins, that become mostly absent from the ciliary axoneme in Zmynd10 mutant respiratory ciliated cells, can sometimes enter the proximal part of the cilium. These results suggest that to a low extent the dynein arms can still assemble and be transported into the cilium in the absence of ZMYND10, thus opening an opportunity for small-molecule therapies that promote protein stability in primary ciliary dyskinesia disease management.

2023-05-16Tese de doutoramento

Acesso aberto

Ver mais

Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

OE

Número da atribuição

SFRH/BD/130272/2017