Background
The molecular identification of neural progenitor cell populations that connect to establish the sympathetic nervous system (SNS) remains unclear. This is due to technical limitations in ...the acquisition and spatial mapping of molecular information to tissue architecture.
Results
To address this, we applied Slide‐seq spatial transcriptomics to intact fresh frozen chick trunk tissue transversely cryo‐sectioned at the developmental stage prior to SNS formation. In parallel, we performed age‐ and location‐matched single cell (sc) RNA‐seq and 10× Genomics Visium to inform our analysis. Downstream bioinformatic analyses led to the unique molecular identification of neural progenitor cells within the peripheral sympathetic ganglia (SG) and spinal cord preganglionic neurons (PGNs). We then successfully applied the HiPlex RNAscope fluorescence in situ hybridization and multispectral confocal microscopy to visualize 12 gene targets in stage‐, age‐ and location‐matched chick trunk tissue sections.
Conclusions
Together, these data demonstrate a robust strategy to acquire and integrate single cell and spatial transcriptomic information, resulting in improved resolution of molecular heterogeneities in complex neural tissue architectures. Successful application of this strategy to the developing SNS provides a roadmap for functional studies of neural connectivity and platform to address complex questions in neural development and regeneration.
Key Findings
We applied Slide‐seq spatial transcriptomics to intact fresh frozen chick trunk tissue.
In parallel, we performed age‐ and location‐matched single cell (sc) RNA‐seq and 10x Genomics Visium to inform our Slide‐seq analysis.
Bioinformatic analyses led to the unique molecular identification of neural progenitor cells within the peripheral sympathetic ganglia (SG) and spinal cord preganglionic neurons (PGNs).
We expanded the HiPlex RNAscope fluorescence in situ hybridization and multispectral confocal microscopy to visualize 12 gene targets in stage‐, age‐ and location‐matched chick trunk tissue sections.
Background
Collective and discrete neural crest cell (NCC) migratory streams are crucial to vertebrate head patterning. However, the factors that confine NCC trajectories and promote collective cell ...migration remain unclear.
Results
Computational simulations predicted that confinement is required only along the initial one‐third of the cranial NCC migratory pathway. This guided our study of Colec12 (Collectin‐12, a transmembrane scavenger receptor C‐type lectin) and Trail (tumor necrosis factor‐related apoptosis‐inducing ligand, CD253) which we show expressed in chick cranial NCC‐free zones. NCC trajectories are confined by Colec12 or Trail protein stripes in vitro and show significant and distinct changes in cell morphology and dynamic migratory characteristics when cocultured with either protein. Gain‐ or loss‐of‐function of either factor or in combination enhanced NCC confinement or diverted cell trajectories as observed in vivo with three‐dimensional confocal microscopy, respectively, resulting in disrupted collective migration.
Conclusions
These data provide evidence for Colec12 and Trail as novel NCC microenvironmental factors playing a role to confine cranial NCC trajectories and promote collective cell migration.
Key Findings
Model simulations predict neural crest cell confinement along the entire length of the migratory domain is not required to maintain discrete streams over long distances.
Colec12 and Trail, we previously identified, confine neural crest cell trajectories off of protein stripes in in vitro stripe assays.
Gain‐ or loss‐of‐function of any single factor or in combinations significantly disrupt the in vivo cranial neural crest migratory pattern.
The neural crest is a migratory population of cells that produces many diverse structures within the embryo. Trunk neural crest cells give rise to such structures as the dorsal root ganglia (DRG) and ...sympathetic ganglia (SG), which form in a metameric pattern along the anterior-posterior axis of the embryo. While static analyses have provided invaluable information concerning the development of these structures, time-lapse imaging of neural crest cells navigating through their normal environment could potentially reveal previously unidentified cellular and molecular interactions integral to DRG and SG development. In this study, we follow fluorescently labeled trunk neural crest cells using a novel sagittal explant and time-lapse confocal microscopy. We show that along their dorsoventral migratory route, trunk neural crest cells are highly motile and interact extensively with neighboring cells and the environment, with many cells migrating in chain-like formations. Surprisingly, the segregated pattern of crest cell streams through the rostral somite is not maintained once these cells arrive alongside the dorsal aorta. Instead, neural crest cells disperse along the ventral outer border of the somite, interacting extensively with each other and their environment via dynamic extension and retraction of filopodia. Discrete sympathetic ganglia arise as a consequence of intermixing and selective reorganization of neural crest cells at the target site. The diverse cell migratory behaviors and active reorganization at the target suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes.
The molecular mechanisms that sort migrating neural crest cells (NCCs) along a shared pathway into two functionally discrete structures, the dorsal root ganglia and sympathetic ganglia (SGs), are ...unknown. We report here that this patterning is attributable in part to differential expression of the chemokine receptor, CXCR4. We show that (1) a distinct subset of ventrally migrating NCCs express CXCR4 and this subset is destined to form the neural core of the sympathetic ganglia, and (2) the CXCR4 ligand, SDF-1, is a chemoattractant for NCCs in vivo and is expressed adjacent to the future SGs. Reduction of CXCR4 expression in NCCs disrupts their migration toward the future SGs, whereas overexpression of CXCR4 in non-SG-destined NCCs induces them to migrate aberrantly toward the SGs. These data are the first to demonstrate a major role for chemotaxis in the patterning of NCC migration and demonstrate the neural crest is composed of molecularly heterogeneous cell populations.
All transiting planet observations are at risk of contamination from nearby, unresolved stars. Blends dilute the transit signal, causing the planet to appear smaller than it really is, or producing a ...false positive detection when the target star is blended with an eclipsing binary. High spatial resolution adaptive optics images are an effective way of resolving most blends. Here we present visual companions and detection limits for 12 Kepler planet candidate host stars, of which 4 have companions within 4". One system (KOI 1537) consists of two similar-magnitude stars separated by 0".1, while KOI 174 has a companion at 0".5. In addition, observations were made of 15 transiting planets that were previously discovered by other surveys. The only companion found within 1" of a known planet is the previously identified companion to WASP-2b. An additional four systems have companions between 1" and 4": HAT-P-30b (3".7, Delta Ks = 2.9), HAT-P-32b (2".9, Delta Ks = 3.4), TrES-1b (2".3, Delta K s = 7.7), and WASP-P-33b (1".9, Delta Ks = 5.5), some of which have not been reported previously. Depending on the spatial resolution of the transit photometry for these systems, these companion stars may require a reassessment of the planetary parameters derived from transit light curves. For all systems observed, we report the limiting magnitudes beyond which additional fainter objects located 0".1-4" from the target may still exist.
We present a large-scale, volume-limited companion survey of 245 late-K to mid-M (K7-M6) dwarfs within 15 pc. Infrared adaptive optics (AO) data were analysed from the Very Large Telescope, Subaru ...Telescope, Canada–France–Hawaii Telescope, and MMT Observatory to detect close companions to the sample from ∼ 1 to 100 au, while digitized wide-field archival plates were searched for wide companions from ∼ 100 to 10 000 au. With sensitivity to the bottom of the main sequence over a separation range of 3 to 10 000 au, multiple AO and wide-field epochs allow us to confirm candidates with common proper motions, minimize background contamination, and enable a measurement of comprehensive binary statistics. We detected 65 comoving stellar companions and find a companion star fraction of 23.5 ± 3.2 per cent over the 3 au to 10 000 au separation range. The companion separation distribution is observed to rise to a higher frequency at smaller separations, peaking at closer separations than measured for more massive primaries. The mass ratio distribution across the q = 0.2–1.0 range is flat, similar to that of multiple systems with solar-type primaries. The characterization of binary and multiple star frequency for low-mass field stars can provide crucial comparisons with star-forming environments and hold implications for the frequency and evolutionary histories of their associated discs and planets.
Human metastatic melanoma cells express a dedifferentiated, plastic phenotype, which may serve as a selective advantage, because melanoma cells invade various microenvironments. Over the last three ...decades, there has been an increased focus on the role of the tumor microenvironment in cancer progression, with the goal of reversing the metastatic phenotype. Here, using an embryonic chick model, we explore the possibility of reverting the metastatic melanoma phenotype to its cell type of origin, the neural-crest-derived melanocyte. GFP-labeled adult human metastatic melanoma cells were transplanted in ovo adjacent to host chick premigratory neural crest cells and analyzed 48 and 96 h after egg reincubation. Interestingly, the transplanted melanoma cells do not form tumors. Instead, we find that transplanted melanoma cells invade surrounding chick tissues in a programmed manner, distributing along host neural-crest-cell migratory pathways. The invading melanoma cells display neural-crest-cell-like morphologies and populate host peripheral structures, including the branchial arches, dorsal root and sympathetic ganglia. Analysis of a melanocyte-specific phenotype marker (MART-1) and a neuronal marker (Tuj1) revealed a subpopulation of melanoma cells that invade the chick periphery and express MART-1 and Tuj1. Our results demonstrate the ability of adult human metastatic melanoma cells to respond to chick embryonic environmental cues, a subset of which may undergo a reprogramming of their metastatic phenotype. This model has the potential to provide insights into the regulation of tumor cell plasticity by an embryonic milieu, which may hold significant therapeutic promise.
All transiting planets are at risk of contamination by blends with nearby, unresolved stars. Blends dilute the transit signal, causing the planet to appear smaller than it really is, or produce a ...false-positive detection when the target star is blended with eclipsing binary stars. This paper reports on high spatial-resolution adaptive optics images of 90 Kepler planetary candidates. Companion stars are detected as close as 0''.1 from the target star. Images were taken in the near-infrared (J and K s bands) with ARIES on the MMT and PHARO on the Palomar Hale 200 inch telescope. Most objects (60%) have at least one star within 6" separation and a magnitude difference of 9. Eighteen objects (20%) have at least one companion within 2" of the target star; six companions (7%) are closer than 075. Most of these companions were previously unknown, and the associated planetary candidates should receive additional scrutiny. Limits are placed on the presence of additional companions for every system observed, which can be used to validate planets statistically using the BLENDER method. Validation is particularly critical for low-mass, potentially Earth-like worlds, which are not detectable with current-generation radial velocity techniques. High-resolution images are thus a crucial component of any transit follow-up program.
Previous studies have suggested that the segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through ...rostral somite halves. We recently showed that the spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. Here, we investigate the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, we measure changes in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a recently developed sagittal slice explant culture and 3D confocal time-lapse imaging. Our results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia.
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