sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

Sox transcription factors are crucial for vertebrate nervous system development. In zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of ventral spinal cord. Our recent study revealed that the loss of sox1a and sox1b function results in a significant decline of V2 subtype neurons (V2s). Using single-cell RNA sequencing, we analyzed the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four time points during embryonic development, employing the Tg(sox1a:eGFP) line. In addition to previously characterized sox1a-expressing neurons, we discovered the expression of sox1a in late-developing intraspinal serotonergic neurons (ISNs). Developmental trajectory analysis suggests that ISNs arise from lateral floor plate (LFP) progenitor cells. Pharmacological inhibition of the Notch signaling pathway revealed its role in negatively regulating LFP progenitor cell differentiation into ISNs. Our findings highlight the zebrafish LFP as a progenitor domain for ISNs, alongside known Kolmer-Agduhr (KA) and V3 interneurons

Neuron-Radial Glial Cell Communication via BMP/Id1 Signaling Is Key to Long-Term Maintenance of the Regenerative Capacity of the Adult Zebrafish Telencephalon

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions

Two plus one is almost three: a fast approximation for multi-view deconvolution

Multi-view deconvolution is a powerful image-processing tool for light sheet fluorescence microscopy, providing isotropic resolution and enhancing the image content. However, performing these calculations on large datasets is computationally demanding and time-consuming even on high-end workstations. Especially in long-time measurements on developing animals, huge amounts of image data are acquired. To keep them manageable, redundancies should be removed right after image acquisition. To this end, we report a fast approximation to three-dimensional multi-view deconvolution, denoted 2D+1D multi-view deconvolution, which is able to keep up with the data flow. It first operates on the two dimensions perpendicular and subsequently on the one parallel to the rotation axis, exploiting the rotational symmetry of the point spread function along the rotation axis. We validated our algorithm and evaluated it quantitatively against two-dimensional and three-dimensional multi-view deconvolution using simulated and real image data. 2D+1D multi-view deconvolution takes similar computation time but performs markedly better than the two-dimensional approximation only. Therefore, it will be most useful for image processing in time-critical applications, where the full 3D multi-view deconvolution cannot keep up with the data flow

Microtome-integrated microscope system for high sensitivity tracking of in-resin fluorescence in blocks and ultrathin sections for correlative microscopy

Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context

Zebrafish biosensor for toxicant induced muscle hyperactivity

Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19^{19}F‐MRI

A labeling strategy for in vivo $^{19}$F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and β-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 $^{19}$F substitutions chemically equivalent, giving rise to a single $^{19}$F-NMR resonance with <10 Hz linewidth. The resulting performance in $^{19}$F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in $^{19}$F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo $^{19}$F-MRI applications