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Ultrastructure of the choroid plexus and its development in the mouse.
The morphogenesis of the choroid plexus of the lateral ventricle of the mouse was studied from the 8th to the 13th post conception day (PCD) in semi- and ultra-thin sections. The choroid plexus appears on PCD 11 with the formation of a horizontal crest in the roof of the interventricular foramen. However, degenerative processes play an important role in the morpho- and histogenetic development of the choroid plexus from PCD 8. During the degenerative period the epithelial cells differentiate and stratify to form the choroid plaque. From PCD 11 the developing choroid plexus expands into the ventricular cavity taking with it mesenchyme and vessels. Ultrastructurally, the epithelial cells differentiate into cells which are more cylindrical and whose nuclei become more elongated. The cytoplasm exhibits numerous prolongations into the ventricular cavity. The golgi complexes, the endoplasmatic reticulum, the ribosomes and polyribosomes are found concentrated in the apical portions of these cells. The apical prolongations of three or more neighbouring cells form "lakes". Junctional complexes between the apical ends of the epithelial cells radiate out from the lakes in which there are microvilli. Macrophages, or "epiplexus cells", frequently give rise to thin cellular processes which encompass protrusions of the epithelial cells. These epiplexus cells contain many vacuoles and phagosomes; numerous mitochondria are localized adjacent to those epiplexus plasma membranes encompassing protrusions. It is proposed that the epithelial cells of the choroid plexus contribute to the formation of the embryonic encephalic liquid.
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Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.
BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.
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The fine structure of the S-100 protein positive cells in the rat pituitary gland: an immunoelectron microscopic study.
We examined the fine structure of rat folliculo-stellate (FS) cells of the anterior pituitary and pituicytes of the neural lobe using immunoelectron microscopy for S-100 protein. The study proves that S-100 protein is solely expressed in the FS cells and pituicytes. However, the ultrastructural differences between these two cell types demonstrated in this study may argue against the hypothesis that the FS cells and the pituicytes have a common origin.
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Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick.
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Human midbrain organoids reveal the characteristics of axonal mitochondria specific to dopaminergic neurons.
Mitochondrial dysfunction and abnormalities in mitochondrial quality control contribute to the development of neurodegenerative diseases. Parkinson's disease is a neurodegenerative disease that causes motor problems mainly due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Axonal mitochondria in neurons reportedly differ in properties and morphologies from mitochondria in somata or dendrites. However, the function and morphology of axonal mitochondria in human dopaminergic neurons remain poorly understood. To define the function and morphology of axonal mitochondria in human dopaminergic neurons, we newly generated tyrosine hydroxylase (TH) reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines from one control and one PRKN-mutant patient iPSC lines and differentiated these iPSC lines into dopaminergic neurons in two-dimensional monolayer cultures or three-dimensional midbrain organoids. Immunostainings with antibodies against axonal and dendritic markers showed that axons could be better distinguished from dendrites of dopaminergic neurons in the peripheral area of three-dimensional midbrain organoids than in two-dimensional monolayers. Live-cell imaging and correlative light-electron microscopy in peripheral areas of midbrain organoids derived from control TH-GFP iPSCs demonstrated that axonal mitochondria in dopaminergic neurons had lower membrane potential and were shorter in length than those in non-dopaminergic neurons. Although the mitochondrial membrane potential did not significantly differ between dopaminergic and non-dopaminergic neurons derived from PRKN-mutant patient lines, these differences tended to be similar to those in control lines. These results were also largely consistent with those of our previous study on somatic mitochondria. The findings of the present study indicate that midbrain organoids are an effective tool to distinguish axonal from dendritic mitochondria in dopaminergic neurons. This may facilitate the analysis of axonal mitochondria to provide further insights into the mechanisms of dopaminergic neuron degeneration in patients with Parkinson's disease.
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Zika viruses encode 5' upstream open reading frames affecting infection of human brain cells.
Zika virus (ZIKV), an emerging mosquito-borne flavivirus, is associated with congenital neurological complications. Here, we investigate potential pathological correlates of virus gene expression in representative ZIKV strains through RNA sequencing and ribosome profiling. In addition to the single long polyprotein found in all flaviviruses, we identify the translation of unrecognised upstream open reading frames (uORFs) in the genomic 5' region. In Asian/American strains, ribosomes translate uORF1 and uORF2, whereas in African strains, the two uORFs are fused into one (African uORF). We use reverse genetics to examine the impact on ZIKV fitness of different uORFs mutant viruses. We find that expression of the African uORF and the Asian/American uORF1 modulates virus growth and tropism in human cortical neurons and cerebral organoids, suggesting a potential role in neurotropism. Although the uORFs are expressed in mosquito cells, we do not see a measurable effect on transmission by the mosquito vector in vivo. The discovery of ZIKV uORFs sheds new light on the infection of the human brain cells by this virus and raises the question of their existence in other neurotropic flaviviruses.
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Tubular arrays in the endoplasmic reticulum in human tumor cells.
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[Relation between the dynamics of early ultrastructural changes in the cerebral cortex of the rat and radiation dose].
The electron microscopic investigation of the sensomotor zone in the cerebral cortex of mature male rats after total neutron and gamma radiation of high doses has demonstrated, that during first three hours after the influence the dynamics of ultrastructural changes in neurons, glial elements and microcirculatory bed depends on the radiation dose. After gamma radiation of 150 Gy, destructive changes in neurons, increased permiability of blood capillaries and development of perivascular edema are noted at earlier time than after radiation dose of 10 Gy. When the greater dose of radiation is used, also earlier reaction of the barrier structures in the brain is observed.
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Human cerebral organoids reveal progenitor pathology in EML1-linked cortical malformation.
Malformations of human cortical development (MCD) can cause severe disabilities. The lack of human-specific models hampers our understanding of the molecular underpinnings of the intricate processes leading to MCD. Here, we use cerebral organoids derived from patients and genome edited-induced pluripotent stem cells to address pathophysiological changes associated with a complex MCD caused by mutations in the echinoderm microtubule-associated protein-like 1 (EML1) gene. EML1-deficient organoids display ectopic neural rosettes at the basal side of the ventricular zone areas and clusters of heterotopic neurons. Single-cell RNA sequencing shows an upregulation of basal radial glial (RG) markers and human-specific extracellular matrix components in the ectopic cell population. Gene ontology and molecular analyses suggest that ectopic progenitor cells originate from perturbed apical RG cell behavior and yes-associated protein 1 (YAP1)-triggered expansion. Our data highlight a progenitor origin of EML1 mutation-induced MCD and provide new mechanistic insight into the human disease pathology.
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Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm.