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Funding Information: Funding: This work was a product of the Project LYSOCIL funded by the European Union Horizon 2020 research and innovation under grant agreement No 811087. It received funding from Fundação para a Ciencia e tecnologia, grant PTDC/BEX-BID/1411/2014; M.R. was funded by the fellowship PD/BD/136927/2018. P.S was funded by the fellowship SFRH/BD/111611/2015; C.B. was funded by the fellowship SFRH/BD/141034/2018; SSL was funded by FCT CEEC-IND 2018. Funding Information: Acknowledgments: The authors want to thank the Fish Facility from NMS. This work was developed with the support of the Fish Facility NMS|FCM that is part of CONGENTO, a Research Infrastructure co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e Tecnologia (Portugal) LISBOA-01-0145-FEDER-022170. A.P. wants to thank the Royal Brompton and Harefield hospital, part of the Guy's and St Thomas' foundation trust, London. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ; Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results. ; publishersversion ; published

This study was supported by the Fundação para a Ciência e a Tecnologia (PTDC/BEXBID/1411/ 2014 research grant). S.S. Lopes was funded by FCT Investigator IF/00951/2012, by NOVA Medical School and by FCT CEEC-IND 2018. P. Sampaio was funded by the PhD fellowship FCT: SFRH/BD/111611/2015. M. Roxo-Rosa was funded by the UID/Multi/04462/2013-LISBOA-01-0145-FEDER-007344 grant (iNOVA4Health). C.M. Quintão was funded by Fundação para a Ciência e Tecnologia (UID/FIS/04559/2013). S.S. Lopes participates in and acknowledge financial support from the COST Action BEAT-PCD (BM1407). S.S. Lopes received funding from project LysoCil funded by the European Union Horizon 2020 research and innovation under grant agreement No 811087. Funding information for this article has been deposited with the Crossref Funder Registry. ; Evaluation of ciliary beat frequency (CBF) performed by high-speed videomicroscopy analysis (HVMA) is one of the techniques required for the correct diagnosis of primary ciliary dyskinesia (PCD). Currently, due to lack of open-source software, this technique is widely performed by visually counting the ciliary beatings per a given time-window. Our aim was to generate open-source, fast and intuitive software for evaluating CBF, validated in Portuguese PCD patients and healthy volunteers. Nasal brushings collected from 17 adult healthy volunteers and 34 PCD-referred subjects were recorded using HVMA. Evaluation of CBF was compared by two different methodologies: the new semi-automated computer software CiliarMove and the manual observation method using slow-motion movies. Clinical history, nasal nitric oxide and transmission electron microscopy were performed for diagnosis of PCD in the patient group. Genetic analysis was performed in a subset (n=8) of suspected PCD patients. The correlation coefficient between the two methods was R-2=0.9895. The interval of CBF values obtained from the healthy control group (n=17) was 6.18-9.17 Hz at 25 degrees C. In the PCD-excluded group (n=16), CBF ranged from 6.84 to 10.93 ...