Molecular Pathways and Animal Models of Defects in Situs.

Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is no less important, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways governing left-right asymmetry are highly conserved and involve multiple components of the TGF-ß superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left, and BMP signaling on the right. In addition, a plethora of other cell signaling pathways including Shh, FGF, and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia that mediate the left-sided propagation of Nodal signaling, followed by left-sided activation of Lefty and then Pitx2, a transcription factor that specifies visceral organ asymmetry. While this overall scheme is well conserved, there are striking species differences, including the finding that motile cilia do not play a role in left-right patterning in some vertebrates. Surprisingly, the direction of heart looping, one of the first signs of organ left-right asymmetry, was recently shown to be specified by intrinsic cell chirality, not Nodal signaling, possibly a reflection of the early origin of Nodal signaling in radially symmetric organisms. How this intrinsic chirality interacts with downstream molecular pathways regulating visceral organ asymmetry will need to be further investigated to elucidate how disturbance in left-right patterning may contribute to complex CHD.

FOXJ1 Variants Causing Primary Ciliary Dyskinesia with Hydrocephalus: A Case Report from Japan.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by motile cilia dysfunction, mostly inherited in an autosomal recessive or X-linked manner. We herein report a 29-year-old woman with PCD caused by a heterozygous frameshift mutation due to a single nucleotide deletion in exon 3 of FOXJ1. Heterozygous de novo mutations in FOXJ1 have been reported as an autosomal-dominant cause of PCD. The patient had situs inversus, congenital heart disease, infertility, and hydrocephalus. However, the nasal nitric oxide level was normal. Long-term macrolide therapy was remarkably effective. This is the first case report of PCD caused by a FOXJ1 variant in Japan.

Novel dominant-negative FOXJ1 mutation in a family with heterotaxy plus mouse model.

Background: Primary ciliary dyskinesia (PCD) is a clinically heterogeneous group of autosomal or, less frequently, X-chromosomal recessive inheritance syndrome of motile cilia dysfunction characterized by neonatal respiratory distress, oto-sino-pulmonary disease, infertility and situs inversus. Recently, type 43 PCD (CILD43, OMIM#618699) was established by autosomal-dominant loss-of-function mutations identified in Forkhead box J1 (FOXJ1). However, the functional validation of FOXJ1 mutations in humans and mice has not been fully performed. Here we studied a three-generation family with heterotaxy and proband with complex congenital heart disease (CHD). Methods: We performed whole-exome sequencing to investigate the causative variant of this family and generated gene knock-in mice carrying the human equivalent mutation by homologous recombination. Then, microscopy analysis was used to characterize the phenotype and ciliary ultrastructure of the model. Effects of the variant on heart anomaly were preliminarily explored through transcriptome sequencing. Results: A novel heterozygous deletion variant (c.1129delC/p.Leu377Trpfs*76) of FOXJ1 was discovered that exerts a dominant-negative effect (DNE) in vitro. Notably, both homozygous (Foxj1c.1129delT/c.1129delT) and heterozygous (Foxj1+/c.1129delT) mice developed situs inversus, hydrocephalus and showed a disruption of trachea cilia structure, whereas these abnormalities were only observed in previously reported Foxj1-/-, not Foxj1+/- mice. Thus, a more severe phenotype and higher expressivity of our mouse model further indicated the DNE of this mutation. Meanwhile, several cardiomyopathy-related genes were differentially expressed in the homozygous Foxj1 knock-in mouse hearts, pointing to a probable function in cardiac pathology. Conclusions: Overall, our study results showed that c.1129delC mutation in FOXJ1 was regarded as the cause of situs inversus in this family and this mutant showed a capacity of DNE over wild-type FOXJ1, causing more serious consequences than the allelic deletion of Foxj1.

Heterozygous FOXJ1 Mutations Cause Incomplete Ependymal Cell Differentiation and Communicating Hydrocephalus.

Heterozygous mutations affecting FOXJ1, a transcription factor governing multiciliated cell development, have been associated with obstructive hydrocephalus in humans. However, factors that disrupt multiciliated ependymal cell function often cause communicating hydrocephalus, raising questions about whether FOXJ1 mutations cause hydrocephalus primarily by blocking cerebrospinal fluid (CSF) flow or by different mechanisms. Here, we show that heterozygous FOXJ1 mutations are also associated with communicating hydrocephalus in humans and cause communicating hydrocephalus in mice. Disruption of one Foxj1 allele in mice leads to incomplete ependymal cell differentiation and communicating hydrocephalus. Mature ependymal cell number and motile cilia number are decreased, and 12% of motile cilia display abnormal axonemes. We observed decreased microtubule attachment to basal bodies, random localization and orientation of basal body patches, loss of planar cell polarity, and a disruption of unidirectional CSF flow. Thus, heterozygous FOXJ1 mutations impair ventricular multiciliated cell differentiation, thereby causing communicating hydrocephalus. CSF flow obstruction may develop secondarily in some patients harboring FOXJ1 mutations. Heterozygous FOXJ1 mutations impair motile cilia structure and basal body alignment, thereby disrupting CSF flow dynamics and causing communicating hydrocephalus.

An RFX transcription factor regulates ciliogenesis in the closest living relatives of animals.

Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S. rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein in vitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.

Cooption of regulatory modules for tektin paralogs during ciliary band formation in a marine annelid larva.

Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.

A novel heterozygous variant of FOXJ1 in a Chinese female with primary ciliary dyskinesia and hydrocephalus: A case report and literature review.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a type of ciliary dyskinesia that is usually caused by autosomal recessive inheritance and can manifest as recurrent respiratory infections, bronchiectasis, infertility, laterality defects, and chronic otolaryngological disease. Although ependymal cilia, which affect the flow of cerebrospinal fluid in the central nervous system, have much in common with respiratory cilia in terms of structure and function, hydrocephalus is rarely associated with PCD. Recently, variants of Forkhead box J1 (FOXJ1) have been found to cause PCD combined with hydrocephalus in a de novo, autosomal dominant inheritance pattern. METHODS: We performed DNA extraction, whole-exome sequencing (WES) analysis, and mutation analysis of FOXJ1 and analyzed the patient's clinical and genetic data. RESULTS: The patient was a 4-year-old female exhibiting normal growth and development. At 3 years and 2 months of age, the patient experienced hand shaking and weakness in the lower limbs. Cardiac ultrasonography showed a right-sided heart, and cranial magnetic resonance imaging showed obstructive hydrocephalus. The nasal nitric oxide level was 54 nL/min. WES indicated a de novo, heterozygous variant of FOXJ1, c.734-735 ins20. This variant was novel, not included in the Human Gene Mutation and Genome Aggregation Database, and likely pathogenic according to the American College of Medical Genetics and Genomics, causing earlier termination of amino acid translation. The patient underwent a neuroendoscopic third ventriculostomy after the diagnosis of obstructive hydrocephalus. Six months after the operation, the patient's motor deficits had improved. CONCLUSION: This is the first report of a de novo, autosomal dominant pattern of FOXJ1 causing PCD combined with hydrocephalus in China. The patient's clinical symptoms were similar to those previously reported. WES confirmed that a novel variant of FOXJ1 was the cause of the PCD combined with hydrocephalus, expanding the spectrum of the genotypes associated with this condition. Physicians should be aware of the correlation of hydrocephalus and PCD and test for FOXJ1 variants.

Seriously cilia: A tiny organelle illuminates evolution, disease, and intercellular communication.

The borders between cell and developmental biology, which have always been permeable, have largely dissolved. One manifestation is the blossoming of cilia biology, with cell and developmental approaches (increasingly complemented by human genetics, structural insights, and computational analysis) fruitfully advancing understanding of this fascinating, multifunctional organelle. The last eukaryotic common ancestor probably possessed a motile cilium, providing evolution with ample opportunity to adapt cilia to many jobs. Over the last decades, we have learned how non-motile, primary cilia play important roles in intercellular communication. Reflecting their diverse motility and signaling functions, compromised cilia cause a diverse range of diseases collectively called "ciliopathies." In this review, we highlight how cilia signal, focusing on how second messengers generated in cilia convey distinct information; how cilia are a potential source of signals to other cells; how evolution may have shaped ciliary function; and how cilia research may address thorny outstanding questions.

Therapeutic strategies to recover ependymal barrier after inflammatory damage: relevance for recovering neurogenesis during development.

The epithelium covering the surfaces of the cerebral ventricular system is known as the ependyma, and is essential for maintaining the physical and functional integrity of the central nervous system. Additionally, the ependyma plays an essential role in neurogenesis, neuroinflammatory modulation and neurodegenerative diseases. Ependyma barrier is severely affected by perinatal hemorrhages and infections that cross the blood brain barrier. The recovery and regeneration of ependyma after damage are key to stabilizing neuroinflammatory and neurodegenerative processes that are critical during early postnatal ages. Unfortunately, there are no effective therapies to regenerate this tissue in human patients. Here, the roles of the ependymal barrier in the context of neurogenesis and homeostasis are reviewed, and future research lines for development of actual therapeutic strategies are discussed.

LRRC6 regulates biogenesis of motile cilia by aiding FOXJ1 translocation into the nucleus.

BACKGROUND: LRRC6 is an assembly factor for dynein arms in the cytoplasm of motile ciliated cells, and when mutated, dynein arm components remained in the cytoplasm. Here, we demonstrate the role of LRRC6 in the active nuclear translocation of FOXJ1, a master regulator for cilia-associated gene transcription. METHODS: We generated Lrrc6 knockout (KO) mice, and we investigated the role of LRRC6 on ciliopathy development by using proteomic, transcriptomic, and immunofluorescence analysis. Experiments on mouse basal cell organoids confirmed the biological relevance of our findings. RESULTS: The absence of LRRC6 in multi-ciliated cells hinders the assembly of ODA and IDA components of cilia; in this study, we showed that the overall expression of proteins related to cilia decreased as well. Expression of cilia-related transcripts, specifically ODA and IDA components, dynein axonemal assembly factors, radial spokes, and central apparatus was lower in Lrrc6 KO mice than in wild-type mice. We demonstrated that FOXJ1 was present in the cytoplasm and translocated into the nucleus when LRRC6 was expressed and that this process was blocked by INI-43, an importin α inhibitor. CONCLUSIONS: Taken together, these results hinted at the LRRC6 transcriptional regulation of cilia-related genes via the nuclear translocation of FOXJ1. Video Abstract.

Ciliated Cells in Ovarian Cancer Decrease with Increasing Tumor Grade and Disease Progression.

Ciliated cell markers expressed in epithelial ovarian cancers (EOC) are associated with improved survival. We examined the distribution of cells expressing ciliated cell markers in various EOC histologies and stages. Immunohistochemistry and/or multiplex immunofluorescence were used to determine the expression of FOXJ1 and/or CAPS (ciliated cell markers) in tissue microarrays including 4 normal fallopian tubes, 6 normal endometria, 16 cystadenomas, 25 borderline tumors, 21 low-grade carcinomas, and 118 high-grade carcinomas (HGSOC) (46 serous, 29 endometrioid, 30 clear cell, 13 mucinous). CAPS+ cells were observed in normal fallopian tubes and endometria and in ~85% of serous benign and borderline tumors and low-grade carcinomas but only in <40% of HGSOC. mRNA data from an independent cohort showed higher FOXJ1 and CAPS expression in serous borderline tumors and low-grade carcinomas compared to HGSOC. In HGSOC, ciliated cell-positive markers were observed in 52% primary tumors compared to 26% of patient-matched synchronous metastases, and 24% metachronous metastases (p = 0.009). mRNA data from an independent HGSOC cohort showed lower levels of CAPS in metastases than in primary tumors (p = 0.03). Overall, the study revealed that ciliated cells were less common in mucinous EOC, the percentage of ciliated cell marker-positive cases decreased with increasing grade, and the percentage of ciliated cells decreased in HGSOC metastases compared to patient-matched primary tumors.

Conditionally reprogrammed asthmatic bronchial epithelial cells express lower FOXJ1 at terminal differentiation and lower IFNs following RV-A1 infection.

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their "parent" pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma (n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs.

FoxJ1 inhibits African swine fever virus replication and viral S273R protein decreases the expression of FoxJ1 to impair its antiviral effect.

African swine fever (ASF) is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar, which is caused by the African swine fever virus (ASFV). ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health. To date, there is no safe and effective commercial vaccine against ASF. Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV. In the present study, RNA-sequencing, RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages (PAMs) infected by ASFV. RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes (ISGs) induced by poly(dA:dT). FoxJ1 revealed a function to positively regulate innate immune response, therefore, suppressing the replication of ASFV. In addition, Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway. Meanwhile, RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1. Altogether, we determined that FoxJ1 plays an antiviral role against ASFV replication, and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1. Our findings provide new insights into the antiviral function of FoxJ1, which might help design antiviral drugs or vaccines against ASFV infection.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

RNA demethylation by FTO stabilizes the FOXJ1 mRNA for proper motile ciliogenesis.

Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here, we report that the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA that encodes the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge, a result owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.

Transcriptional regulation of multiciliated cell differentiation.

Multiciliated cells (MCC) project dozens to hundreds of motile cilia from the cell surface to generate fluid flow across epithelial surfaces or turbulence to promote the transport of gametes. The MCC differentiation program is initiated by GEMC1 and MCIDAS, members of the geminin family, that activate key transcription factors, including p73 and FOXJ1, to control the multiciliogenesis program. To support the generation of multiple motile cilia, MCCs must undergo massive centriole amplification to generate a sufficient number of basal bodies (modified centrioles). This transcriptional program involves the generation of deuterosomes, unique structures that act as platforms to regulate centriole amplification, the reactivation of cell cycle programs to control centriole amplification and release, and extensive remodeling of the cytoskeleton. This review will focus on providing an overview of the transcriptional regulation of MCCs and its connection to key processes, in addition to highlighting exciting recent developments and open questions in the field.

Consecutive Hypoxia Decreases Expression of NOTCH3, HEY1, CC10, and FOXJ1 via NKX2-1 Downregulation and Intermittent Hypoxia-Reoxygenation Increases Expression of BMP4, NOTCH1, MKI67, OCT4, and MUC5AC via HIF1A Upregulation in Human Bronchial Epithelial Cells.

Previous studies have shown that the experimental models of hypoxia-reoxygenation (H/R) mimics the physiological conditions of ischemia-reperfusion and induce oxidative stress and injury in various types of organs, tissues, and cells, both in vivo and in vitro, including human lung adenocarcinoma epithelial cells. Nonetheless, it had not been reported whether H/R affected proliferation, apoptosis, and expression of stem/progenitor cell markers in the bronchial epithelial cells. In this study, we investigated differential effects of consecutive hypoxia and intermittent 24/24-h cycles of H/R on human bronchial epithelial (HBE) cells derived from the same-race and age-matched healthy subjects (i.e., NHBE) and subjects with chronic obstructive pulmonary disease (COPD) (i.e., DHBE). To analyze gene/protein expression during differentiation, both the NHBE and DHBE cells at the 2nd passage were cultured at the air-liquid interface (ALI) in the differentiation medium under normoxia for 3 days, followed by either culturing under hypoxia (1% O2) for consecutively 9 days and then returning to normoxia for another 9 days, or culturing under 24/24-h cycles of H/R (i.e., 24 h of 1% O2 followed by 24 h of 21% O2, repetitively) for 18 days in total, so that all differentiating HBE cells were exposed to hypoxia for a total of 9 days. In both the normal and diseased HBE cells, intermittent H/R significantly increased HIF1A, BMP4, NOTCH1, MKI67, OCT4, and MUC5AC expression, while consecutive hypoxia significantly decreased NKX2-1, NOTCH3, HEY1, CC10, and FOXJ1 expression. Inhibition of HIF1A or NKX2-1 expression by siRNA transfection respectively decreased BMP4/NOTCH1/MKI67/OCT4/MUC5AC and NOTCH3/HEY1/CC10/FOXJ1 expression in the HBE cells cultured under intermittent H/R to the same levels under normoxia. Overexpression of NKX2-1 via cDNA transfection caused more than 2.8-fold increases in NOTCH3, HEY1, and FOXJ1 mRNA levels in the HBE cells cultured under consecutive hypoxia compared to the levels under normoxia. Taken together, our results show for the first time that consecutive hypoxia decreased expression of the co-regulated gene module NOTCH3/HEY1/CC10 and the ciliogenesis-inducing transcription factor gene FOXJ1 via NKX2-1 mRNA downregulation, while intermittent H/R increased expression of the co-regulated gene module BMP4/NOTCH1/MKI67/OCT4 and the predominant airway mucin gene MUC5AC via HIF1A mRNA upregulation.

Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating.

STUDY QUESTION: How does exposure to a testosterone rich environment affect the function and gene expression of human fallopian tube epithelium (hFTE)? SUMMARY ANSWER: Elevated testosterone level alters several gene transcripts that regulate cilia expression and negatively impacts the rate of cilia beating. WHAT IS KNOWN ALREADY: The presence of estrogen in the follicular phase of the menstrual cycle increases the human fallopian tube ciliary beating frequency. The luteal phase, triggered by ovulation and increasing progesterone, is marked by a decrease in ciliary beating. Women with polycystic ovarian syndrome (PCOS) may have twice the serum level of testosterone than ovulatory women. To date, the effect of elevated androgens on the function of the human fallopian tube is not well-understood. We chose to examine the impact of elevated testosterone on hFTE. STUDY DESIGN, SIZE, DURATION: A prospective basic science study of human fallopian tube specimens from reproductive-aged women undergoing benign gynecologic surgery was performed. Fallopian tube removal at a large US academic center was collected and provided to us to continue with epithelium isolation and culturing. A total of 12 patients were analyzed in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fallopian tube epithelium was isolated and exposed to two different conditions: normal with low testosterone concentration of 0.8 nM and PCOS-like, with high testosterone concentration of 2 nM. The study was conducted in both static and dynamic conditions in microfluidic devices for a total of 14 days, after which the tissue was collected for processing including RNA extraction, quantitative PCR and immunohistochemistry. After the first 7 days of each experiment, a sample of tissue from each condition was imaged to quantify cilia beating frequency. MAIN RESULTS AND THE ROLE OF CHANCE: hFTE exposed to the 2 nM testosterone displayed slower cilia beating, inhibited estrogen signaling and decreased expression of the ciliary marker FOXJ1 when compared to stimulation with 0.8 nM testosterone. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The in vivo response to elevated testosterone may differ from in vitro studies. RNA amount was limited from tissue cultured in the microfluidic devices as compared to static culture. WIDER IMPLICATIONS OF THE FINDINGS: Understanding elevated testosterone in tubal function may explain an additional contribution to subfertility in women with PCOS and other hyper-androgen disorders, aside from oligo-ovulation. Furthermore, this adds to the body of literature of fallopian tube function using a microfluidic device. STUDY FUNDING/COMPETING INTEREST(S): NIH grants: UH3 ES029073 and R01 CA240301. There are no competing interests.

The role of motile cilia in the development and physiology of the nervous system.

Motile cilia are miniature, whip-like organelles whose beating generates a directional fluid flow. The flow generated by ciliated epithelia is a subject of great interest, as defective ciliary motility results in severe human diseases called motile ciliopathies. Despite the abundance of motile cilia in diverse organs including the nervous system, their role in organ development and homeostasis remains poorly understood. Recently, much progress has been made regarding the identity of motile ciliated cells and the role of motile-cilia-mediated flow in the development and physiology of the nervous system. In this review, we will discuss these recent advances from sensory organs, specifically the nose and the ear, to the spinal cord and brain ventricles. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.