Development Of Ciliated Cells Research Articles

Emx2 is an essential regulator of ciliated cell development across embryonic tissues

Emx2 is an essential regulator of ciliated cell development across embryonic tissues

Uncovering the Ciliogenesis Regulatory Network

Ciliopathies comprise disorders associated with aberrant function of essential organelles called cilia. Cilia are found in almost all vertebrate cells, with a subset of this population being motile and/or multiciliated. Over 1,000 proteins have been associated with the ciliary proteome, yet the regulatory circuit controlling the formation of cilia remains lesser known. Our lab has identified transcription factors and coactivators that contribute to the ciliogenesis circuit. Such factors include orphan nuclear receptor Esrrγ and coactivator Ppargc1a/PGC1a. Independently, these factors regulate both mono‐ and multi‐ciliated cells in the early signaling node, ear, and embryonic kidney (pronephros). Absence of these factors results in decreased multiciliated cells, decreased cilia length, and increased unciliated basal bodies. Considering these phenotypic similarities, we hypothesized a cooperative relationship between Esrrγ and Pppargc1a in the context of ciliated cell development. By modelling dual heterozygosity with genetic interaction studies, we found that ppargc1aworks synergistically with esrrγato regulate prostaglandin signaling to support ciliary outgrowth and multiciliated cell fate choice. While previous studies have alluded to the interaction of Esrrγ and Ppargc1a/PGC1a in metabolism, this is the first report of a cooperative effect that is essential for ciliogenesis or lineage decisions in development. This study has far‐reaching implications, as cilia are present in various tissues, and these factors may serve as therapeutic targets for ciliopathic conditions and birth defects.

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

SummaryMotile 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.

Ppargc1a Controls Ciliated Cell Development by Regulating Prostaglandin Biosynthesis

SUMMARYCilia are microtubule-based organelles that function in a multitude of physiological contexts to perform chemosensing, mechanosensing, and fluid propulsion. The process of ciliogenesis is highly regulated, and disruptions result in disease states termed ciliopathies. Here, we report that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a) is essential for ciliogenesis in nodal, mono-, and multiciliated cells (MCCs) and for discernment of renal tubule ciliated cell fate during embryogenesis. ppargc1a performs these functions by affecting prostaglandin signaling, whereby cilia formation and renal MCC fate are restored with prostaglandin E2 (PGE2) treatment in ppargc1a-deficient animals. Genetic disruption of ppargc1a specifically reduces expression of the prostanoid biosynthesis gene prostaglandin-endoperoxide synthase 1 (ptgs1), and suboptimal knockdown of both genes shows this synergistic effect. Furthermore, ptgs1 overexpression rescues ciliogenesis and renal MCCs in ppargc1a-deficient embryos. These findings position Ppargc1a as a key genetic regulator of prostaglandin signaling during ciliated cell ontogeny.

Ppargc1a Controls Ciliated Cell Development by Regulating Prostaglandin Biosynthesis

Cilia are microtubule-based organelles that function in a multitude of physiological contexts to perform chemosensing, mechanosensing, or fluid propulsion. The process of ciliogenesis is highly regulated and disruptions result in disease states termed ciliopathies. Here, we show novel roles for peroxisome proliferator-activated receptor gamma 1 alpha ( ppargc1a ) during ciliogenesis in nodal, mono-, and multiciliated cells (MCCs) as well as discernment of renal tubule ciliated cell fate during embryogenesis. We discovered that ppargc1a performs both roles by affecting prostaglandin signaling, where cilia formation and renal MCC fate were restored with prostaglandin E 2 (PGE 2 ) treatment. Genetic disruption of ppargc1a specifically reduced expression of the prostanoid biosynthesis gene prostaglandin-endoperoxide synthase 1 ( ptgs1 ), and suboptimal knockdown of both genes revealed a synergistic effect. Further, ptgs1 overexpression rescued ciliogenesis and renal MCCs in ppargc1a deficient embryos. These findings position Ppargc1a as an essential genetic regulator of prostaglandin signaling during ciliated cell ontogeny.

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus.

BackgroundMicroRNAs (miRNAs) are important post-transcriptional regulators which control growth and development in eukaryotes. The cestode Echinococcus granulosus has a complex life-cycle involving different development stages but the mechanisms underpinning this development, including the involvement of miRNAs, remain unknown.ResultsUsing Illumina next generation sequencing technology, we sequenced at the genome-wide level three small RNA populations from the adult, protoscolex and cyst membrane of E. granulosus. A total of 94 pre-miRNA candidates (coding 91 mature miRNAs and 39 miRNA stars) were in silico predicted. Through comparison of expression profiles, we found 42 mature miRNAs and 23 miRNA stars expressed with different patterns in the three life stages examined. Furthermore, considering both the previously reported and newly predicted miRNAs, 25 conserved miRNAs families were identified in the E. granulosus genome. Comparing the presence or absence of these miRNA families with the free-living Schmidtea mediterranea, we found 13 conserved miRNAs are lost in E. granulosus, most of which are tissue-specific and involved in the development of ciliated cells, the gut and sensory organs. Finally, GO enrichment analysis of the differentially expressed miRNAs and their potential targets indicated that they may be involved in bi-directional development, nutrient metabolism and nervous system development in E. granulosus.ConclusionsThis study has, for the first time, provided a comprehensive description of the different expression patterns of miRNAs in three distinct life cycle stages of E. granulosus. The analysis supports earlier suggestions that the loss of miRNAs in the Platyhelminths might be related to morphological simplification. These results may help in the exploration of the mechanism of interaction between this parasitic worm and its definitive and intermediate hosts, providing information that can be used to develop new interventions and therapeutics for the control of cystic echinococcosis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-736) contains supplementary material, which is available to authorized users.

Zygotic Porcn Paternal Allele Deletion in Mice to Model Human Focal Dermal Hypoplasia

In mouse and humans, the X-chromosomal Porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands, thus representing a bottleneck in the secretion of Wnt ligands. In humans, mutations in PORCN cause the X-linked dominant syndrome Focal Dermal Hypoplasia (FDH, OMIM#305600). This disorder is characterized by ecto-mesodermal dysplasias and shows a highly variable phenotype, potentially due to individual X chromosome inactivation patterns. To improve the understanding of human FDH, we have established a mouse model by generation of Porcn heterozygous animals carrying a zygotic deletion of the paternal allele. We show that heterozygous female fetuses display variable defects that do not significantly affect survival in the uterus, but lead to perinatal lethality in more than 95% of females. Rare survivors develop to adulthood and display variable skeletal and skin defects, representing an adult zygotic mouse model for human FDH. Although not frequently reported in humans, we also observed bronchopneumonia, rhinitis, and otitis media in these animals, suggesting a potential link between Porcn function and the normal development of ciliated cells in these tissues.

Ciliated Cells Differentiated from Mouse Embryonic Stem Cells

In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

Ciliated-cell differentiation and gene expression

Ciliated cells play an integral role in the defense mechanisms of the respiratory system. By the coordinated beating of their cilia they provide the force necessary to clear potentially harmful material from the airways. We have been investigating the regulation of ciliated-cell differentiation and gene expression. Using a culture system that allows us to positively or negatively regulate the development of the ciliated-cell phenotype, we have previously reported that the expression of axonemal dynein heavy chain mRNAs are regulated in parallel with the development of ciliated cells. To identify other genes important to the development or function of ciliated cells, differential display was used to compare mRNA isolated from cultures of ciliated or nonciliated rat tracheal epithelial cells. Two novel genes, KPL1 and KPL2, have been identified whose expression is increased in parallel with ciliated-cell differentiation. Two transcripts of KPL1 are expressed in a tissue-specific pattern; KPL1 is particularly highly expressed in brain. The sequence of KPL1 predicts a 188 or 223 amino acid protein which contains a pleckstrin homology domain. Pleckstrin homology domains have been shown to bind inositolphosphates and G-proteins and function as signal-dependent membrane adapters. KPL1 therefore may function in a signal transduction pathway important to the development or maintenance of the ciliated-cell phenotype. KPL2 shows more limited distribution and is predominantly expressed in tissues which contain axonemes. KPL2 is predicted to encode a 1744 amino acid protein which contains many functional motifs, including nuclear localization signals, an ATP-binding domain, a proline-rich region, and a calponin homology domain. KPL2 may thus be involved in transmitting signals to the nucleus during ciliated-cell differentiation.

Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry.

Winged helix transcription factors play important roles in cellular differentiation and cell-specific gene expression. To define the role of the winged helix factor hepatocyte nuclear factor/forkhead homologue (HFH)-4, a targeted mutation was created in the mouse hfh-4 gene. No expression of HFH-4 was detected in hfh-4(-)/- mice by RNA blot analysis, in situ hybridization, or RT-PCR. hfh-4(-)/- mice were noted to have abnormalities of organ situs consistent with random determination of left-right asymmetry. In addition, a complete absence of cilia was noted in hfh-4(-)/- mice. The hfh-4 gene is thus essential for nonrandom determination of left-right asymmetry and development of ciliated cells. Homozygous mutant mice also exhibited prenatal and postnatal growth failure, perinatal lethality and, in some cases, hydrocephalus. RT-PCR revealed an absence of left-right dynein (lrd) expression in the embryonic lungs of hfh-4(-)/- mice, suggesting that HFH-4 may act by regulating expression of members of the dynein family of genes. The abnormalities in ciliary development and organ situs in hfh-4(-)/- mice are similar to those observed in human congenital syndromes such as Kartagener syndrome. Targeted mutation of hfh-4 thus provides a model for elucidating the mechanisms regulating ciliary development and determination of left-right asymmetry.

Health Effects of Sulphur-Related Environmental Air Pollution—The Role of Acidic Aerosols

The contribution of gaseous and particulate pollutants to the ambient air pollution mixture varies with time and geographic location. Often sulphur dioxide and particles are considered together because they are the primary products of fossil fuel combustion processes and are usually present together as components of a complex mixture. These air pollution scenarios can be simulated by considering mixtures of gaseous sulphur IV (sulphur dioxide), particle-associated sulphur IV (neutral sulphite particles) and particle-associated hydrogen ions. In a first attempt to study respiratory responses induced by sulphur-related air pollution, dogs were exposed to gaseous and particulate sulphur IV over a period of 290 days (Heyder et al., 1992). No indication of chronic bronchitis was observed, but there were functional and morphometrical indications of early stages of lung emphysema, hyperplastic changes in the nasal cavity and disturbances of ciliated cell development in the trachea (Tables 1 and 2). A more complex air pollution mixture was used in the study presented here. Over a period of 13 months particle-associated hydrogen ions were delivered to canine lungs in addition to gaseous and particulate sulphur IV. Since the size distributions of particles carrying H and sulphur IV were almost identical and since particles carrying H grew in the intrapulmonary moist air almost as much as particles carrying sulphur IV, the intrapulmonary sites of H and sulphur IV deposition was very similar. Therefore, in all likelihood interactions between respiratory responses induced by sulphur IV and H occurred and synergy was anticipated.

Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

Regulation of mucociliary differentiation of rat tracheal epithelial cells by type I collagen gel substratum.

Adult rat tracheal epithelial cells plated at low density on type I collagen gel-coated permeable membranes in air-liquid interface cultures rapidly proliferate to reach high cell densities and display a pseudostratified mucociliary epithelium after 2 wk in culture. To determine the importance of exogenous extracellular matrix for RTE cell growth and differentiation, RTE cells were seeded on uncoated or type I collagen gel-coated membranes. Growth rate and plateau cell densities were similar under these two conditions; however, RTE cell attachment was twofold higher on type I collagen gel-coated membranes. Cell distribution during log growth was significantly different, with numerous small colonies observed on Giemsastained uncoated membranes and fewer, larger colonies observed on type I collagen gel-coated membranes. Under both conditions, the epithelial morphology was predominantly pseudostratified in plateau-phase cultures. Based on experiments measuring the percentage of secretory cells and the amount of mucous secretion, development of the secretory cell phenotype was delayed in the absence of exogenous matrix but reached similar levels of secretory differentiation by day 13 of culture. In contrast, the development of ciliated cells was markedly reduced on uncoated membranes. These data suggest that although plating cells on type I collagen gel enhances cell attachment and accelerates the onset of secretory differentiation, type I collagen gel seems to be more critical for ciliated cell differentiation than for secretory cell differentiation.

Regulation of ciliated cell differentiation in cultures of rat tracheal epithelial cells.

The cellular pathway of ciliated cell differentiation and its regulation is poorly defined. To begin to understand the process of ciliated cell differentiation, we sought to identify factors regulating ciliated cell development in vitro. Rat tracheal epithelial (RTE) cells were cultured on collagen gel-coated membranes at an air-liquid interface in hormone- and growth factor-supplemented medium (complete medium [CM]). Under these conditions, RTE cells first proliferate and then differentiate into a pseudostratified mucociliary epithelium. Ciliated cell differentiation was measured using a monoclonal antibody, RTE3, which was shown to specifically react with the plasma membrane of ciliated cells. Cultures were immunostained in situ, and the percentage of the culture surface covered with ciliated cells was estimated using videomicroscopy and an image analysis program. If an air-liquid interface was not created and the cells were maintained in the submerged state, ciliated cell differentiation was suppressed 25-fold. Culture in the absence of mitogenic components present in CM, including epidermal growth factor (EGF), cholera toxin (CT), or bovine pituitary extract, resulted in 2- to 4-fold increases in the percentage of ciliated cells. When both EGF and CT were removed from the media, DNA synthesis and total cell number was reduced, while ciliated cell differentiation increased as much as 5-fold. These results demonstrate that submersion inhibits, while withdrawal of mitogenic compounds promotes, ciliated cell differentiation in vitro.

Chapter 10 Ciliogenesis of Rat Tracheal Epithelial Cells in Vitro

Publisher Summary To facilitate the studies of the factors and processes regulating the differentiation and function of ciliated cells of the airways, methods for culturing dissociated airway epithelial cells that promote de novo ciliogenesis in vitro have been developed for hamster, rat, dog, guinea pig, and human respiratory cells. Studies have demonstrated the importance of a preformed extracellular matrix and/or culture on permeable supports at an air-liquid interface for the development of ciliated cells. This chapter describes the procedures used in developing an in vitro system that supports proliferation and then mucociliary differentiation of dissociated primary rat tracheal epithelial (RTE) cells. Under isolation and culture of cells, the chapter discusses solutions and materials and procedures. Cannulas, used for the isolation of RTE cells, are made from 5-cm pieces of polyethylene tubing. One end of the tubing is pressed against a heated scalpel blade to form a slight flange. These are stored in 70% ethanol. There are details on the basic and complete mediums, bovine pituitary extract and tissue culture insert. The procedure describes the isolation of rat tracheal epithelial cells, plating and growth of cells, differentiation of rat tracheal epithelial cells, and quantitation of ciliated cell differentiation. Ciliated cells are visible in the viable cultures by phase microscopy. To estimate the extent of ciliated cell differentiation in large numbers of cultures, an immunostaining procedure was developed using a monoclonal antibody, RTE-3, which reacts specifically with ciliated cells. RTE-3 can also be used to identify ciliated cells in cytospins prepared from dissociated cultures. If some of the growth-stimulating compounds are removed from the medium after the cells have reached confluence (day 7), the number of ciliated cells present at day14 can be two- to fourfold higher than that obtained when the cells are cultured with complete medium.

Inhibition of ciliated cell differentiation by fluid submersion.

Rat tracheal epithelial (RTE) cells, plated at low density on collagen gel-coated membranes, differentiate into a mucociliary epithelium when cultured at an air-liquid interface (ALI). However, when RTE cells are cultured submerged in media, ciliated cell differentiation is drastically reduced. This study examined possible mechanisms for the inhibition of ciliated cell differentiation by submersion. Ciliated cell differentiation was measured using a monoclonal antibody specific for rat ciliated cells. Removing growth stimulatory compounds from both the basal and apical media increased ciliated cell differentiation in submerged cultures, indicating that submersion inhibits, but does not prevent, ciliogenesis. However, the effect of submersion was independent of the composition of the apical media. The depth of apical fluid was important, with depths > or = 1 mm causing almost complete inhibition of ciliated cell differentiation, while a depth of 0.5 mm allowed significant ciliogenesis. Submersion appeared to block ciliated cell differentiation at an early step, because ciliated cell development required several days following creation of an ALI. Once ciliogenesis was initiated in ALI cultures, submersion did not reverse or inhibit the development off ciliated cells. These studies have provided new information on the inhibition of ciliated cell differentiation by fluid submersion.

Ultrastructural observations of the ciliated cyst in the human uterine tube epithelium

The ciliated cysts have been identified as a common manner of development of ciliated cells through light microscopic studies, however recent ultrastructural studies have Reported that they should be regarded as an expression of atypical ciliogenesis. In the present study we attempt to elucidate the detailed structure of the ciliated cysts and their significance in ciliogenesis in the human uterine tube epithelium.Human uterine tubes were obtained from 20 women who underwent total hysterectomies due to gynecological diseases. The ampullar segments were cut into small pieces, and fixed in 2.5% glutaraldehyde-2% paraformaldehyde (1/2 Kamovsky) in a 0.1M phosphate buffer solution (pH 7.4). These specimens were postfixed in a 1% Osmium tetroxide solution (Millonig), dehydrated with a graded series of ethanol and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a transmission electron microscope.The ciliated cysts were detected as a round or oval intracytoplasmic cavity provided with many ciliary apparatuses and microvilli (Fig. 1).

Long-term exposure of dogs to a sulphite aerosol: IV. Effects on extrapulmonary airway morphology

The extrapulmonary airways of 8 male beagle dogs were examined histopathologically after continuous exposure to a sulphite aerosol at a concentration of 1 mg/m 3 for 290 days. Hyperplastic foci were observed in the respiratory region of the posterior nasal cavity. The changes were characterized by a thickened epithelial layer resulting from epithelial proliferation, by a loss of secretory material and by moderate mononuclear cell infiltration. In the larynx an increased number of non-ciliated cells, and slight mononuclear cell infiltration were found. Tracheal changes were limited to an increased number of non-ciliated cells in the membranous portion. TEM (transmission electron microscopy) showed the changes in the trachea to be caused by disorder in the development of ciliated cells. These findings suggest that sulphite aerosols have adverse effects on the extrapulmonary airways of beagle dogs.

Histological effects of vitamin A on limb regeneration in the larval axolotl, Ambystoma mexicanum

Vitamin A causes profound changes in the development of pattern during amphibian limb regeneration. Vitamin A effects include the induction of duplications of skeletal structures in the anteroposterior, proximodistal, and dorsoventral axes. The purpose of this investigation was to study the underlying histological changes that are induced in the regenerating limb of the larval axolotl, Ambystoma mexicanum, by treatment with vitamin A. Axolotl larvae (7–10 cm in length) had forelimbs amputated through the radius and ulna and were then immersed in 75 IU/mL retinol palmitate for 14 days. Limbs were removed and fixed at intervals over the period of regeneration, both during and beyond the period of retinoid treatment. They were then examined in the light microscope, scanning electron microscope, or transmission electron microscope. Compared with the controls, the retinol palmitate treated regenerating limbs exhibited the development of an eccentric epidermal cap which was always displaced towards the posterior edge of the limb. Beneath this epidermal cap, the density of the cells of the blastema was greater than that of the cells towards the anterior edge of the developing blastema where the cells were much less densely arranged. Epidermal changes induced by retinol palmitate included the development of a very uneven and creviced surface, with a great deal of variation in cell size, and the development of ciliated cells in the surface layer of the epidermis. The significance of these observations for pattern modification by vitamin A are discussed.