Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelium, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.     

Development of the brain: a vital role for cerebrospinal fluid

There has been considerable recent progress in understanding the processes involved in brain development. An analysis of a number of neurological conditions, together with our studies of the hydrocephalic Texas (H-Tx) rat, presents an important role for cerebrospinal fluid (CSF) in the developmental process. The fluid flow is essentially one-way and the location of the choroid plexuses in the lateral, third, and fourth ventricles allows for the possibility of new components being added to the fluid at these points. The role of the fourth ventricular CSF is particularly interesting since this is added to the fluid downstream of the cerebral hemisphere germinal epithelium (the main site of cortical cell proliferation and differentiation) and is destined for the basal cisterns and subarachnoid space suggesting different target cells to those within the ventricular system. Moreover, other sources of additions to the CSF exist, notably the subcommissural organ, which sits at the opening of the third ventricle into the cerebral aqueduct and is the source of Reisner's fibre, glycoproteins, and unknown soluble proteins. In this paper a model for the role of CSF is developed from studies of the development of the cortex of the H-Tx rat. We propose that CSF is vital in controlling development of the nervous system along the whole length of the neural tube and that the externalisation of CSF during development is essential for the formation of the layers of neurones in the cerebral cortex.     

Surface of the vascular plexuses of human cerebral ventricles

The racemose part of vascular plexuses of the lateral ventricles has been studied in fetuses and persons at various age in the scanning and transmissive electron microscopes. In the magistral arteries of the vascular plexuses the neural trunks have been studied. They form the periadventitial plexus with a more complex network of connections in mature persons. The sulci and the elongated folds between them make the relief of the plexus. A rough surface of epitheliocytes has deep craters and irregular protrusions, microvilli, cilia and spherical bodies. The epitheliocytes are arranged in a single layer and connected with each other by means of protoplasmic peduncles. Over the epithelial layer, as single groups, Kolmer cells are situated.     

The effects of high-sucrose diets and of maternal diabetes on the ultrastructure of the visceral yolk sac endoderm in rat embryos developing in vivo and in vitro

The ultrastructure of the visceral yolk sac endoderm of in vivo developing 9- to 13-day-old embryos from 2 diabetic rat models (streptozotocin diabetes and Cohen--genetically determined--diabetes) and from nondiabetic rats fed high sucrose diets have been studied. This was compared to yolk sacs from 9.5-day-old embryos cultured for 48 h in sera from diabetic and nondiabetic rats fed a high-sucrose diet. Light-microscopic, TEM and SEM studies showed that the pathological cellular changes in the visceral yolk sac endoderm from diabetic rats were first observed on day 9 and were most severe among 11-day-old embryos. In vitro culture of control rat embryos in serum from experimental animals induced a reduction in the number of microvilli, of vacuolar intracellular inclusions and an increase in the number of degenerated endodermal cells. SEM studies showed that in addition to disappearance of microvilli, the majority of cells were collapsed and had degenerated cell membranes. Culture of embryos from diabetic animals in control serum only slightly reversed the pathological changes in the visceral yolk sac endoderm. A good correlation exists between the rate of embryonic malformations in diabetic rats and an index of endodermal-cell damage in the visceral yolk sac.     

Multiple Primary Cilia Modulate the Fluid Transcytosis in Choroid Plexus Epithelium

Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral‐to‐apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production.     

Development and differentiation of the brain ventricular system in tadpoles of Xenopus laeris (Daudin) (Amphibia, Anura)

The development and the differentiation of the ventricular system of the brain of tadpoles of the South African Clawed Toad, Xenopus laevis (Daudin), is studied by light microscopy (stages 45 to 66) and scanning and transmission electron microscopy (stages 50 to 66). Special interest is paid to the ependymal structures of the foramen of Monroe, the ventricles of the diencephalon, the mesencephalon, and the rhombencephalon, and to the ependymal of the central canal and the choroid plexus of the third and fourth ventricle. At early developmental stages the lower two thirds of the ventricles are dominated by blebs, cytoplasmatic protrusions of the ependymal cells. During the development they become reduced and replaced by cilia. The number of cilia and microvilli increases strongly towards the end of the metamorphosis. The surface structures demonstrated by scanning electron microscopy are discussed in respect to morphology and physiology.     

Effects of pulsing electromagnetic fields on the prenatal and postnatal development in mice and rats: in vivo and in vitro studies

Electromagnetic fields (EMF) might have various biological effects on the developing embryo. We studied the effects of pulsing electromagnetic fields (PEMF) on the in vitro development of preimplantation mouse embryos and of early somite rat embryos as well as on the in vivo development of rat embryos. We used PEMF at frequencies of 1, 20, 50, 70, and 100 Hz with a tension of 0.6 V/m. The embryos were exposed to PEMF throughout the experimental period. PEMF at frequencies of 20 and 50 Hz were embryotoxic, inhibiting over 50% of blastocysts from hatching and further development, all within 72 h of culture. PEMF at frequencies of 50 and 70 Hz induced 22% and 30% incidence of malformations in 10.5 day old rat embryos after 48 h in culture. The main malformations were absence of telencephalic, optic, and otic vesicles and of forelimb buds. In addition, retarded growth and development manifested by fewer somites, reduction in crown-rump length, and retarded closure of the neural tube were found in many embryos. No significant pathological changes were found by TEM in PEMF-exposed embryos. Disappearance of microvilli and collapse of apical parts of endodermal cells were observed by SEM in many yolk sacs of embryos exposed to 50 and 70 Hz PEMF. A slightly reduced litter average, a reduction or increase of weight, and a delay in eye opening was observed among offspring of pregnant rats exposed throughout pregnancy to PEMF at frequencies of 20, 50, and 100 Hz. No malformations were observed among these offspring. The mechanism of PEMF-induced embryotoxicity and teratogeneity is unknown, as is the mechanism of the "protective effects" of the mother on the rat embryos exposed to PEMF in vivo.     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.     

Correlative scanning-transmission electron microscopic examination of the perinatal rat brain

Summary The ultrastructural organization of the perinatal hypothalamus and the dynamics of neuronal and ependymal growth and plasticity were examined in this investigation. The brains of fetal rats 16, 17 and 18 days in utero and those of postnatal rats 1–16 days post partum were fixed with aldehyde fixatives and prepared for combined SEM/TEM analysis. By day 17 in utero the ventricular (ependymal) surfaces of the fetal thalamic wall, cerebral vesicle and rhomboid fossa were relatively well differentiated with cilia and microvilli. Type II histiocytes were the first supraependymal cell to appear upon the ventricular lumen and were evident by day 17 in utero. In contrast, the apical surfaces of tanycytes of the infundibular recess as well as those of most other circumventricular organs were poorly differentiated and unremarkable. Tanycytes of the infundibular recess exhibited a simple hexagonal mosaic pattern of apposed plasmalemmata and even by day 1 post partum few cilia or microvilli were evident.By day 5–6 post partum Type I supraependymal neurons and axonal processes began to make their appearance with some emerging from the underlying parenchyma of the median eminence. By day 16 post partum the ventricular surface of the infundibular recess was comparable with that of the adult.The Type I supraependymal neurons are remarkably similar in their ultrastructural organization with parvicellular neurosecretory neurons elsewhere in the endocrine hypothalamus. Their emergence at day 5–6 post partum suggests a possible correlation with the critical period of sexual differentiation and a potential receptor role for this cell line. On the contrary this phenomenon may simply be a developmental anomaly. Nonetheless, the mergence of such elements upon the lumen of the third cerebral ventricle underscores a remarkable degree of neuronal plasticity in the perinatal hypothalamus.Supported by USPHS Program Project Grant NS 11642-04 and USPHS-BRSG Grant RR-05403.The authors wish to thank N. Kutryeff for her excellent technical assistance     

CHANGES IN CELL SURFACE MORPHOLOGY DURING DIFFERENTIATION OF MICROMERE- AND MESOMERE-DERIVED CELLS OF THE SEA URCHIN EMBRYO

Micromeres and mesomeres isolated from 16-cell embryos of the sea urchin, Strongylocentrotus intermedius , were cultured in vitro , and changes in the cells surface architecture during the differentiation of the micromere- and mesomere-derived cells were observed using scanning electron microscopy. Two types of the distribution of the surface microvilli were observed in both blastomere-derived cell masses. One type showed a uniform distribution of the microvilli and the other type showed an uneven one. Though many microvilli were observed in most of both mesomere and micromere-derived cells at the 64-cell stage and the early blastula stage (16 hr after the 16-cell stage at 6°C) respectively, the microvilli decreased in number at the later stages in both blastomere-derived cell masses as compared with the 64-cell stage and the early blastula stage respectively. Rapid disappearance of the surface microvilli was observed in the micromere-derived cells in contrast with the mesomere-derived cells which still had many microvilli even at the midmesenchyme stage.     

Changes in cell surface primary cilia and microvilli concurrent with measurements of fluid flow across the rabbit corneal endothelium ex vivo

Primary cilia and microvilli have been reported on the mammalian rabbit corneal endothelium but their relationship to cell function is undefined. Six corneas from healthy 2 kg female albino rabbits were glutaraldehyde-fixed post mortem (15:00 h) or twelve corneal stroma-endothelial preparations incubated at 37 degrees C under an applied hydrostatic pressure of 20 cm H2O for 4 h prior to fixation. The corneal endothelium was assessed by quantitative scanning electron microscopy. Cells fixed immediately post mortem were decorated with small stubby microvilli (average 21 +/- 13/100 micron 2), and only 25% of the cells were decorated with primary cilia having an average length of 2.44 +/- 1.56 microns. Following 4 h ex vivo incubation with a phosphate-buffered Ringer solution, conspicuous microvilli developed to an average density of 40 +/- 19/100 micron 2 and primary cilia were found on 12% of the cells, having on average length of 2.27 +/- 1.38 microns. Following 4 h incubation in a bicarbonate-buffered Ringer solution, small stubby microvilli developed to a density of 49 +/- 18/100 micron 2, and 40% of the cells showed primary cilia with an average length of 4.31 +/- 1.93 microns; the net trans-endothelial fluid flow in the latter set was 60% greater. These studies indicate that the primary cilia on corneal endothelial cells might be responsive to fluid flow, but that mild mechanical and/or chemical stress could also be the cause of the change since the elaboration of primary cilia can be accompanied by microvilli as well.     

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.     

Effect of oestradiol-17 beta and progesterone on oviductal transport and early development of mouse embryos

Transport of embryos through the oviduct, cleavage rate and transformation of morulae to blastocysts, were delayed in females ovariectomized on Day 2 of pregnancy. Oestradiol-17 beta in doses of 60 to 6000 pg/day for 3 days did not normalize the transport of embryos, but the transformation of morulae to blastocysts reached values near or equal to those of the controls, in spite of a lowered rate of cleavage. Progesterone at a dose of 100 micrograms/day, resulted in normal transport, rate of cleavage and rate of differentiation. Treatment with both hormones had synergistic effects on transport and the rate of cleavage and differentiation. These results give further support to the concept that ovarian hormones are the controlling factors for these processes in early pregnancy.     

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.     

Early morphogenesis of ciliated cells in human oral cavity

Ciliated cells were found in the epithelium of the oral cavity of human embryos and fetuses starting from the seventh week of prenatal development. At the early stages of prenatal development (until the 13th week), cells with cilia cover most of the dorsal surface of the tongue and the soft palate, whereas they are found only near the gland ducts in the circumvallate and foliate lingual papillae after 17 weeks of development. The ultrastructure of the axoneme of cilia corresponds to the structure of motile cilia and is represented by nine microtubule doublets that surround the central pair of microtubule singlets. An immunohistochemical study performed on weeks 10–12 of development identified nerve endings associated with the ciliated cells. Until the 14th week of development, the cytoplasm of ciliated cells is immunopositive for NSE. The spatial distribution of ciliated cells in the tongue epithelium until the 13th week of development is not related to the morphogenesis of lingual papillae, and their role in the human oral cavity during the first trimester of pregnancy is unclear and requires further study.     

Effect of bacterial endotoxin on migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis

The effect of bacterial lipopolysaccharide endotoxin (LPS), immune system activator, on differentiation and migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis has been studied. Intraperitoneal introduction of LPS (18 jg/kg) to pregnant rats on the 12th day of pregnancy led to 50% decrease in total number of GRH-neurons in the forebrain of 17-day-old embryos and 17% decrease in 19-day-old embryos. At the same time, the number of GRH-neurons in the nasal area of the head of 17- and 19-day-old embryos increased by 40 and 50%, respectively, whereas it increased by 20% in olfactory bulbs of 17-day-old embryos and did not changed in olfactory bulbs of 19-day-old embryos. Neither the total number of neurons nor their distribution patterns were affected by the introduction of LPS into pregnant rats on the 15th day of pregnancy. Singular localization of GRH-neurons in embryo forebrain was observed after LPS administration, whereas the neurons were located by groups of 3-4 cells in rostral areas. Therefore, at the early stages of pregnancy, LPS was shown to suppress initial stages of differentiation and migration of GRH producing neurons. The effects observed in our study may be mediated by LPS-induced, proinflammatory cytokines.     

Effect of bacterial endotoxin on migration of gonadotropin-releasing hormone-producing neurons in rat embryogenesis

The effect of bacterial lipopolysaccharide endotoxin (LPS), immune system activator, on differentiation and migration of gonadotropin-releasing hormone-producing neurons in rat embryogenesis has been studied. Intraperitoneal injection with LPS (18 μg/kg) into pregnant rats on the 12th day of pregnancy led to 50% decrease in total number of GnRH-neurons in the forebrain of 17-day-old embryos and 17% decrease in 19-day-old embryos. At the same time, the number of GnRH-neurons in the nasal area of the head of 17- and 19-day-old embryos was increased by 40 and 50%, respectively, whereas it increased by 20% in olfactory bulbs of 17-day-old embryos and was not changed in olfactory bulbs of 19-day-old embryos. Neither the total number of neurons nor their distribution patterns were affected by LPS injection into pregnant rats on the 15th day of pregnancy. Singular localization of GnRH-neurons in embryo forebrain was observed after LPS administration, whereas the neurons were located by groups of 3–4 cells in rostral areas. Therefore, at the early stages of pregnancy, LPS was shown to suppress initial stages of differentiation and migration of GnRH-producing neurons. The effects observed in our study may be mediated by LPS-induced proinflammatory cytokines.     

Development of muscles in the dorsal foreleg of rat embryos. A light microscopic study with the in situ cholinesterase staining technique

The development of muscles from the dorsal side of the forelegs from 13- to 21-day-old rat embryos was investigated under a light microscope. The muscle blastemata and individual muscles were stained in situ with the cholinesterase technique. The first muscle blastemata are visible on the early day 13. It appears that mainly myotubes are stained. The antebrachial and brachial extensor muscles form separated anlagen which connect on the late day 13 in the proximal region of the extensor carpi ulnaris muscle. The individualization of the muscles in a muscle blastema takes place on days 13 and 14. On day 15 all extensor muscles are visible. However, at this time the inserting points of some of these muscles are not yet visible after staining with alcian blue. On the early day 16 the motor end-plates are conspicuous. Due to the content of unspecific cholinesterase in rat embryos the tendons are also stained on day 16. Muscles and tendons remain stainable until birth. In addition to the muscles also the nerves, especially the epifascial nerves, stain very well with the acetylcholinesterase reaction.