Stellaris Dlc Activator
Sanny Olafeso
Stochastic Optical Reconstruction Microscopy (STORM) reconstructs a super-resolution fluorescence image by combining precise localization information for individual fluorophores in complex fluorescent specimens. N-STORM takes advantage of Nikon's powerful Ti2-Eclipse inverted microscope and applies high-accuracy, multi-color localization and reconstruction in three dimensions (xyz) to enable super-resolution imaging at tenfold the resolution of conventional light microscopes (up to approximately 20 nm in xy).
Tenfold improvement of axial resolution up to 50nm: In addition to lateral super-resolution, N-STORM utilizes proprietary methods to achieve a tenfold enhancement in axial resolution over conventional light microscopes and provide nanoscale information in 3D. The 3D-Stack function allows multiple 3D STORM images from different Z positions to be captured and stitched into one image to create thicker STORM images. Tenfold improvement of lateral resolution up to 20nm: N-STORM utilizes high accuracy localization information for thousands of individual fluorophores present in a field of view to create breathtaking "super-resolution" images, exhibiting spatial resolution that is 10 times greater than conventional optical microscopes. Dynamic super-resolution imaging: Newly developed optics and illumination systems, optimized for sCMOS technology, have increased image acquisition speeds by up to 10 times. With acquisition times reduced from minutes to seconds*, dynamic events in live specimens can now be captured with molecular level resolution. Multi-color imaging capability: Multi-color super-resolution imaging can be carried out using both activator-reporter pairs for sequential activation imaging and activator-free labels for continuous activation imaging. This flexibility allows users to easily gain critical insights into the localization and interaction properties of multiple proteins at the molecular level. High definition, high density images: Newly developed excitation optics and improved image acquisition rates provide increased molecule localization density, resulting in clearer images of macromolecular structures. Large image acquisition area: New intermediate zoom lenses in the imaging system have been developed and optimized for a wide field of view. The wide-view mode achieves 80 μm x 80 μm, a 4-fold increase in imaging area compared to previous models. Seamless switching between imaging modalities for multi-scale experiments: The N-STORM can be simultaneously combined with a confocal microscope such as the A1+. A desired location in a sample can be specified in a low-magnification/large FOV confocal image and acquired in super-resolution by simply switching the imaging method. Combining a confocal microscope with a super-resolution system can provide a method for gaining larger contextual views of the super-resolution information. High Power Oil immersion objectives: These objectives provide the high numerical apertures required for N-STORM imaging. The HP objectives are compatible with the ultrahigh power lasers required for inducing rapid photoswitching of fluorophores. They provide improved axial chromatic aberration correction to achieve the highest level of precision in localization and image alignment for 3D multi-color STORM imaging. The AC-type objectives that support the Auto Correction Collar of the Ti2-E microscope allow precise and easy adjustment of the correction collar.
Spatiotemporally precise and robust cell fate transitions, which depend on specific signaling cues, are fundamental to the development of appropriately patterned tissues. The fidelity and precision with which photoreceptor fates are recruited in the Drosophila eye exemplifies these principles. The fly eye consists of a highly ordered array of 750 ommatidia, each of which contains eight distinct photoreceptors, R1-R8, specified sequentially in a precise spatial pattern. Recruitment of R1-R7 fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. Here we ask how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. We find that during R2-R5 specification PntP2 is coexpressed with a closely related but previously uncharacterized isoform, PntP3. Using CRISPR/Cas9-generated isoform specific null alleles we show that under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and that the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, our data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.
Properly patterned tissues require cells transit from a multipotent state to diverse differentiated states in a precise spatiotemporal manner. Meanwhile the programs that direct cell fate adoption must be reliable despite genetic and nongenetic variation. In this study we use the Drosophila photoreceptors as a model system for understanding how specific and robust cell fate transitions are achieved. The specification of seven distinct photoreceptors R1-R7 requires repetitive inductive signaling from the receptor tyrosine kinase (RTK)/ mitogen-activated protein kinase (MAPK). The transcription factor Pointed (Pnt) operates downstream of MAPK to initiate the changes in gene expression appropriate to the particular transition. We asked how the generic MAPK/Pnt signal contributes to different photoreceptor fates. Previous work showed that R1-R7 photoreceptor specification can be subdivided into two rounds that experience different RTK signaling strengths. We find distinct Pnt regulatory networks operate in the two rounds, with the first round network incorporating a novel unstudied Pnt isoform, PntP3. Its inclusion stabilizes developmental transitions when signaling is reduced. We compare and contrast the expression patterns and transactivation potentials of the Pnt isoforms and uncover a web of transcriptional cross-regulation between them. Based on these explorations, we propose that the use of distinct Pnt network topologies provides an adaptive mechanism that permits reliable cell fate transitions under different MAPK signaling environments.
Copyright: 2020 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work, and C.W, JF.B.L, M.Z.L and I.R, were supported by grants from the National Institutes of Health (R01-GM080372 to I.R. and R01 EY025957 to I.R.). Further research support was from a National Institutes of Health grant P30 CA014599 to the University of Chicago Cancer Center. The University of Chicago provided additional salary support to I.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Specification of all photoreceptors except R8 requires inductive signaling by the receptor tyrosine kinase (RTK) / Ras / mitogen-activated protein kinases (MAPK) pathway via the transcriptional effector Pointed (Pnt), the Drosophila homologue of the mammalian ETS family activators ETS1 and ETS2 [12,13]. Multipotent retinal progenitors must therefore translate this generic RTK/Pnt signal into specific photoreceptor fates. Numerous studies have focused on combinatorial regulation to integrate the inputs from RTK/Pnt with specific inputs from regionally expressed transcription factors and other signaling pathway effectors. For example, RTK/Pnt, the Spalt transcription factors and Notch signaling collectively specify R4 fates in the first round [14,15] whereas in the second round, RTK/Pnt and Notch signaling integrate with a different transcription factor, Lozenge, to regulate prospero transcription and R7 fates [16,17].
Increasing the complexity of these combinatorial codes, RTK signaling inputs are not identical during the two rounds of specification. Fate specification in the first round relies exclusively on signaling initiated by the epidermal growth factor receptor (EGFR), while specification of R1, R6 and R7 second round fates involves a second RTK, Sevenless (Sev) in addition to EGFR [18,19]. Although only R7 fates are lost in a sev mutant, the R1 and R6 precursors express Sev, physically contact the Boss ligand-expressing R8 cell, and so are likely to have active Sev signaling [20]. Because both EGFR and Sev use the same Ras/MAPK/Pnt signaling cascade, it has been proposed that cells specified in the second round experience stronger MAPK activation than those in the first round [12,21]. How the Pnt response is tailored to these two different signaling environments has not been explored.
A previous sequential activation model posited that transient RTK/MAPK signaling activates PntP2, which in turn activates pntP1 transcription, and that PntP1 then provides a stable, signaling-independent, transcriptional input to the combinatorial codes that initiate the specification of R1-R7 photoreceptor fates (Fig 1C; [25]). However the expression pattern of PntP2 suggests further complexity, with lower levels in the region of R2-R5 specification and then higher levels in more posterior regions where R1, R6, R7 and cone fates are recruited [25]. These differences parallel the differences in RTK signaling in the two rounds of photoreceptor specification and motivated us to explore how the Pnt response is tuned to these two distinct signaling environments.