Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rar alpha, and rar beta) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgf beta 2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rar beta, meis2, hoxb5, tgf beta 2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgf beta 2, and id2 spatial distribution; to increase rar beta, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.The authors would like to thank Ana Lima for slide sectioning and Rita Lopes for contributing to the initiation of this project. This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Caracterização da via de sinalização Hippo no desenvolvimento pulmonar da galinha

Dissertação de mestrado em Ciências da SaúdeHIPPO signalling pathway, and its effector YAP, have been recognized as important growth regulators during embryonic development, adult tissue regeneration, homeostasis, and tumorigenesis. YAP phosphorylation status impacts the transcription of HIPPO target genes responsible for conveying HIPPO response. Curiously, the inhibition of this pathway in the liver causes overgrowth, whereas in pancreas hypoplasia, therefore suggesting phenotypic differences depending on the organ system. Nevertheless, the information regarding pulmonary development remains limited. Therefore, this project aims to determine HIPPO’s role during pulmonary branching and, for the first time, in the avian animal model. All experiments were conducted in embryonic chick lungs during early branching stages. The spatial distribution of HIPPO signalling members was characterized by in situ hybridization. Moreover, in vitro lung explant culture was performed for 48 hours, and protein levels of phosphorylated-YAP (pYAP)/YAP were evaluated by Western blot at two time-points (0 and 48 hours). Additionally, lung explants were cultured with verteporfin (VP) (5, 7.5 and 10 µM), which prevents YAP-TEAD binding, resulting in supressed transcription of target genes (ctgf). Subsequently, explants were also subjected to in situ hybridization for ctgf, to confirm successful HIPPO modulation. In addition, explants were morphometrically analysed to determine the impact of Hippo modulation on lung growth. Finally, lung explants were evaluated for cytotoxicity by LDH assay. In situ hybridization revealed that all HIPPO signalling members are present in the developing lung, throughout early stages of chick lung branching, except for mst2 and lats2. The western blot analysis revealed similar expression levels of both YAP and pYAP in the three stages studied. After 48 hours in culture, YAP and pYAP protein levels were slightly decreased when compared to 0 hours, nonetheless, the pYAP/YAP ratio was maintained. Lung explants treated with 7.5 and 10 µM of VP displayed statistically significant overall reduction in lung size, when compared to controls. VP-treated lung explants displayed a decrease in ctgf expression, in a dose-dependent manner, thus confirming alteration in HIPPO signalling. Lastly, the LDH assay revealed no cellular toxicity in all verteporfin conditions. This study demonstrates, for the first time, the presence of HIPPO signalling in early stages of avian pulmonary branching. Gene/protein expression profile and pathway modulation studies indicate that HIPPO is active and possibly involved in lung growth control.A via de sinalização HIPPO, e seu efetor YAP, têm um papel importante na regulação do crescimento durante o desenvolvimento embrionário, regeneração de tecidos no adulto, homeostasia e tumorigénese. O estado de fosforilação da proteína YAP tem impacto na transcrição de genes alvo específicos, responsáveis por transmitir a resposta da via. Curiosamente, a inibição da via HIPPO no fígado causa crescimento, enquanto no pâncreas induz hipoplasia, sugerindo diferenças fenotípicas dependendo do órgão. No entanto, a informação disponível sobre o desenvolvimento pulmonar é ainda limitada. Assim, este projeto como objetivo determinar o papel da via HIPPO durante a ramificação pulmonar e, pela primeira vez, no modelo de galinha. Neste sentido, utilizaram-se pulmões embrionários de galinha nos estadios iniciais de ramificação. O padrão de expressão dos componentes da via HIPPO foi caracterizado por hibridização in situ. Paralelamente, realizaram-se cultura de explantes pulmonares in vitro, durante 48 horas, e os níveis proteicos de YAP e YAP fosforilado (pYAP) foram avaliados por Western blot às 0 e 48 horas. Mais ainda, os explantes pulmonares foram tratados com verteporfina (5, 7,5 e 10 µM) que impede a ligação de YAP TEAD resultando na supressão da transcrição de genes alvo (ctgf). Subsequentemente, os explantes foram analisados por hibridação in situ para ctgf, para confirmar a modulação da via HIPPO. Os explantes foram também analisados morfometricamente para determinar o impacto da modulação desta via no crescimento pulmonar. Finalmente, os explantes pulmonares foram avaliados quanto à citotoxicidade pelo ensaio de LDH. A hibridização in situ revelou que todos os componentes da via HIPPO estão presentes no pulmão em desenvolvimento, nos estadios iniciais da ramificação do pulmão de galinha, com exceção de mst2 e lats2. Por outro lado, a análise de Western blot revelou níveis de expressão semelhantes de YAP e pYAP nos estadios estudados. Após 48 horas, os níveis de proteína YAP e pYAP diminuíram quando comparados a 0 horas, no entanto, a relação pYAP/YAP manteve-se constante. Os explantes de pulmão tratados com 7,5 e 10 µM de verteporfina (VP) apresentaram uma redução global, e estatisticamente significativa, no tamanho do pulmão, quando comparados com o controlo. Os explantes de pulmão tratados com verteporfina exibiram uma diminuição na expressão de ctgf, dependente da dose, confirmando assim a alteração na sinalização de HIPPO. Por fim, o ensaio de LDH não revelou toxicidade celular nas condições de verteporfina testadas. Este estudo demonstra, pela primeira vez, a presença da via de sinalização de HIPPO nos estadios iniciais da ramificação pulmonar de galinha. O perfil de expressão bem como os estudos de modulação da via sugerem que a via HIPPO está ativa e possivelmente envolvida no controlo do crescimento pulmonar, em galinha.The work presented in this dissertation was performed at the Surgical Sciences Research Domain of the Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal (ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal). The work was supported by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

DataSheet1_Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection.docx

One of the most prevalent healthcare-associated infection is the urinary tract infection (UTI), caused by opportunistic pathogens such as Candida albicans or non-albicans Candida species (NACS). Urine culture methods are routinely used for UTI diagnostics due to their specificity, sensitivity and low-cost. However, these methods are also laborious, time- and reagent-consuming. Therefore, diagnostic methods relying on nucleic acids have been suggested as alternatives. Nucleic acid-based methods can provide results within 24 h and can be adapted to point-of-care (POC) detection. Here, we propose to combine fluorescence in situ hybridization (FISH) with a microfluidic platform for the detection of Candida spp. As a case study we used C. tropicalis, which is reported as the second most common NACS urine isolate obtained from patients suspected with UTI. The microfluidic platform proposed in this study relies on hydrodynamic trapping, and uses physical barriers (e.g., microposts) for the separation of target cells from the suspension. Using a specific peptide nucleic acid (PNA) probe, the FISH procedure was applied onto previously trapped C. tropicalis cells present inside the microfluidic platform. Fluorescence signal intensity of hybridized cells was captured directly under the epifluorescence microscope. Overall, the PNA probe successfully detected C. tropicalis in pure culture and artificial urine (AU) using FISH combined with the microfluidic platform. Our findings reveal that FISH using nucleic acid mimics (PNA) in combination with microfluidics is a reliable method for the detection of microorganisms such as C. tropicalis. As such, this work provides the basis for the development of a POC detection platform in the future.</p