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Cell Feb 2023Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the...
Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.
Topics: Animals; Humans; Body Patterning; Embryonic Development; Gene Expression Regulation, Developmental; Mammals; Mesoderm; Morphogenesis; Somites; Wnt Signaling Pathway; Organoids
PubMed: 36657441
DOI: 10.1016/j.cell.2022.12.042 -
Cell Feb 2023The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tail bud give rise to...
The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tail bud give rise to tissues that generate spinal cord, skeleton, and musculature. This raises the question of how the embryo achieves axial elongation and patterning. While ethics necessitate in vitro studies, the variability of organoid systems has hindered mechanistic insights. Here, we developed a bioengineering and machine learning framework that optimizes organoid symmetry breaking by tuning their spatial coupling. This framework enabled reproducible generation of axially elongating organoids, each possessing a tail bud and neural tube. We discovered that an excitable system composed of WNT/FGF signaling drives elongation by inducing a neuromesodermal progenitor-like signaling center. We discovered that instabilities in the excitable system are suppressed by secreted WNT inhibitors. Absence of these inhibitors led to ectopic tail buds and branches. Our results identify mechanisms governing stable human axial elongation.
Topics: Humans; Body Patterning; Mesoderm; Wnt Signaling Pathway; Embryo, Mammalian; Organoids
PubMed: 36657443
DOI: 10.1016/j.cell.2022.12.043 -
Acta Crystallographica. Section E,... Jan 2013In the title complex, [Mg(C44H28N4)(C6H4N2)(H2O)], the Mg(2+) cation is octa-hedrally coordinated and lies on an inversion center with the axially located...
In the title complex, [Mg(C44H28N4)(C6H4N2)(H2O)], the Mg(2+) cation is octa-hedrally coordinated and lies on an inversion center with the axially located 4-cyano-pyridine and aqua ligands exhibiting 50% substitutional disorder. The cyano-bound 4-cyano-pyridine mol-ecule also is disordered across the inversion centre. The four N atoms of the pyrrole rings of the dianionic 5,10,15,20-tetra-phenyl-porphyrin ligand occupy the equatorial sites of the octa-hedron [Mg-N = 2.0552 (10) and 2.0678 (11) Å] and the axial Mg-(N,O) bond length is 2.3798 (12) Å. The crystal packing is stabilized by weak inter-molecular C-H⋯π inter-actions.
PubMed: 23476319
DOI: 10.1107/S1600536812049434 -
Heliyon Feb 2023To examine histologic characteristics of macular Bruchś membrane defects (BMD) in axially elongated eyes.
PURPOSE
To examine histologic characteristics of macular Bruchś membrane defects (BMD) in axially elongated eyes.
DESIGN
Histomorphometric study.
METHODS
Using light microscopy, we examined enucleated human globes for BMDs.
RESULTS
In 247 eyes, BMDs were detected in 15 (6.1%) eyes (axial length:27.0-36.0 mm), in 10 of them in the macular region. Prevalence and size of BMDs (mean:1.93 ± 1.62 mm; range:0.22mm-6.24 mm) correlated with longer axial length (OR:1.52; 95%CI:1.19,1.94; P = 0.001) and higher prevalence of scleral staphylomas (OR:16.3; 95%CI:2.67,99.3; P < 0.001). The BMDs were smaller than corresponding gaps in the retinal pigment epithelium (RPE) (1.93 ± 1.62 mm versus 2.61 mm ± 1.73 mm; P = 0.003), and larger than corresponding gaps in the inner nuclear layer (0.43 ± 0.76 mm; P = 0.008) and inner limiting membrane bridges (0.13 ± 0.33 mm; P = 0.001). Choriocapillaris thickness, BM thickness and RPE cell density did not vary (all P > 0.05) between the BDM border and adjacent areas. In the BMD, choriocapillaris and RPE were absent. The sclera was thinner in the BDM area than in adjacent areas (0.28 ± 0.19 mm versus 0.36 ± 0.13 mm; P = 0.006).
CONCLUSIONS
BMDs as hallmarks of myopic macular degeneration are characterized by longer gaps in the RPE and smaller gaps in the outer nuclear layer and inner nuclear layer, by localized scleral thinning, and by a spatial association with scleral staphylomas. Thickness of the choriocapillaris and density of the RPE cell layer, both absent within the BDMs, do not vary between the BMD border and adjacent regions. The results suggest an association between BDMs and absolute scotomas, stretching of the adjacent retinal nerve fiver layer, and an axial elongation-associated stretching effect on BM as etiology of the BDMs.
PubMed: 36793950
DOI: 10.1016/j.heliyon.2023.e13257 -
Light, Science & Applications 2016Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion...
Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.
PubMed: 27398242
DOI: 10.1038/lsa.2016.134 -
Acta Crystallographica. Section E,... Jul 2011In the title compound, C(31)H(27)N(3)O(2), the two piperidine rings fused to each other each adopt a slightly distorted chair conformation. The phenyl rings on the...
In the title compound, C(31)H(27)N(3)O(2), the two piperidine rings fused to each other each adopt a slightly distorted chair conformation. The phenyl rings on the N-unsubstituted piperidine ring occupy an equatorial position, while those on the N-nitroso-substituted piperidine ring are in axial positions. The NO group is approximately coplanar with the piperidine ring with a maximum deviation of 0.048 (4) Å. The dihedral angles between the mean planes of the axially and equatorially oriented phenyl rings are 27.7 (1) and 31.9 (1)°, respectively. Mol-ecular packing is stabilized by weak inter-molecular C-H⋯O and C-H⋯π inter-actions.
PubMed: 21837083
DOI: 10.1107/S1600536811022203 -
Nature Communications Aug 2022Light sheet microscopes reduce phototoxicity and background and improve imaging speed compared to widefield and confocal microscopes. However, when equipped with...
Light sheet microscopes reduce phototoxicity and background and improve imaging speed compared to widefield and confocal microscopes. However, when equipped with Gaussian beams, the axial resolving power of a light sheet microscope and the observable field of view are inversely related. Light sheets based on dithered optical lattices improve axial resolution and beam uniformity compared Gaussian beams by using axially structured illumination patterns. However, these advantages come at the expense of an increased total illumination to the specimen and a decreased axial confinement of the illumination pattern. Using simulations and experimental measurements in fixed and live cells, we quantify the differences between Gaussian and lattice light sheets on beam uniformity, axial resolution, lateral resolution, and photobleaching. We demonstrate how different optical lattice illumination patterns can be tuned to prioritize either axial resolution or optical sectioning. Finally, we introduce an approach to spectrally fuse sequential acquisitions of different lattice light sheet patterns with complementary optical properties to achieve both high resolution and low background images.
Topics: Microscopy; Normal Distribution; Photobleaching
PubMed: 35941165
DOI: 10.1038/s41467-022-32341-w -
BMC Biology Nov 2023Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for the high-throughput cellular imaging of optically cleared, large-sized specimens,...
BACKGROUND
Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for the high-throughput cellular imaging of optically cleared, large-sized specimens, such as the brain. Despite the development of various OT-LSM techniques, achieving submicron resolution in all dimensions remains.
RESULTS
We developed a high-resolution open-top axially swept LSM (HR-OTAS-LSM) for high-throughput and high-resolution imaging in all dimensions. High axial and lateral resolutions were achieved by using an aberration-corrected axially swept excitation light sheet in the illumination arm and a high numerical aperture (NA) immersion objective lens in the imaging arm, respectively. The high-resolution, high-throughput visualization of neuronal networks in mouse brain and retina specimens validated the performance of HR-OTAS-LSM.
CONCLUSIONS
The proposed HR-OTAS-LSM method represents a significant advancement in the high-resolution mapping of cellular networks in biological systems such as the brain and retina.
Topics: Mice; Animals; Microscopy, Fluorescence; Brain; Neurons
PubMed: 37940973
DOI: 10.1186/s12915-023-01747-3 -
Asia-Pacific Journal of Ophthalmology... 2019The process of emmetropization is the adjustment of the length of the optical axis to the given optical properties of the cornea and lens after the end of the second... (Review)
Review
The process of emmetropization is the adjustment of the length of the optical axis to the given optical properties of the cornea and lens after the end of the second year of life. Up to the end of the second year of life, the eye grows spherically. Axial elongation in the process of emmetropization after the second year of life is associated with a thinning of the retina and a reduced density of retinal pigment epithelium (RPE) cells in the equatorial and retroequatorial region, and a thinning of the choroid and sclera, starting at the equator and being most marked at the posterior pole. In contrast, retinal thickness and RPE density in the macular region and thickness of Bruch membrane (BM) in any region are independent of axial length. It led to the hypothesis that axial elongation occurs by the production of additional BM in the equatorial and retroequatorial region leading to a decreased RPE density and retinal thinning in that region and a more tube-like than spherical enlargement of the globe, without compromise in the density of the macular RPE cells and in macular retinal thickness. The increased disc-fovea distance in axially myopic eyes is caused by the development and enlargement of parapapillary, BM-free, gamma zone, whereas the length of macular BM, and indirectly macular RPE cell density, and macular retinal thickness, remain constant.
Topics: Axial Length, Eye; Bruch Membrane; Disease Progression; Humans; Macula Lutea; Myopia; Optic Disk
PubMed: 31425168
DOI: 10.1097/01.APO.0000578944.25956.8b -
Philosophical Transactions of the Royal... Nov 2022Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated...
Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.
Topics: Animals; Carbon Fiber; Mice; Microscopy, Atomic Force; Myocytes, Cardiac
PubMed: 36189808
DOI: 10.1098/rstb.2021.0326