The Warburg effect is necessary to promote glycosylation in the blastema during zebrafish tail regeneration.

Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-ß signaling and blocking glucose metabolism largely mimicked inhibition of TGF-ß receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

High-precision registration between zebrafish brain atlases using symmetric diffeomorphic normalization.

Atlases provide a framework for spatially mapping information from diverse sources into a common reference space. Specifically, brain atlases allow annotation of gene expression, cell morphology, connectivity, and activity. In larval zebrafish, advances in genetics, imaging, and computational methods now allow the collection of such information brain-wide. However, due to technical considerations, disparate datasets may use different references and may not be aligned to the same coordinate space. Two recent larval zebrafish atlases exemplify this problem: Z-Brain, containing gene expression, neural activity, and neuroanatomical segmentations, was acquired using immunohistochemical stains, while the Zebrafish Brain Browser (ZBB) was constructed from live scans of fluorescent reporters in transgenic larvae. Although different references were used, the atlases included several common transgenic patterns that provide potential "bridges" for transforming each into the other's coordinate space. We tested multiple bridging channels and registration algorithms and found that the symmetric diffeomorphic normalization algorithm improved live brain registration precision while better preserving cell morphology than B-spline-based registrations. Symmetric diffeomorphic normalization also corrected for tissue distortion introduced during fixation. Multi-reference channel optimization provided a transformation that enabled Z-Brain and ZBB to be co-aligned with precision of approximately a single cell diameter and minimal perturbation of cell and tissue morphology. Finally, we developed software to visualize brain regions in 3 dimensions, including a virtual reality neuroanatomy explorer. This study demonstrates the feasibility of integrating whole brain datasets, despite disparate reference templates and acquisition protocols, when sufficient information is present for bridging. Increased accuracy and interoperability of zebrafish digital brain atlases will facilitate neurobiological studies.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

In toto imaging of the migrating Zebrafish lateral line primordium at single cell resolution.

The zebrafish Posterior Lateral Line primordium (PLLp) has emerged as an important model system for studying many aspects of development, including cell migration, cell type specification and tissue morphogenesis. Despite this, basic aspects of PLLp biology remain incompletely understood. The PLLp is a group of approximately 140 cells which pioneers the formation of the Posterior Lateral Line (LL) system by migrating along the length of the embryo, periodically depositing clusters of epithelial cells, which will go on to form the mature sense organs of the lateral line, called neuromasts. The neuromasts are formed within the migrating PLLp as protoneuromasts: the first protoneuromast is formed close to the trailing end and additional protoneuromasts are formed sequentially, progressively closer to the leading edge of the migrating collective. We imaged the migration of PLL primordia and tracked every cell in the lateral line system over the course of migration. From this data set we unambiguously determined the lineage and fate of every cell deposited by the migrating PLLp. We show that, on average, proliferation across the entire PLLp is weakly patterned, with leading cells tending to divide more slowly than trailing cells. Neuromasts are formed sequentially by local expansion of existing cells along the length of the PLLp, not by self-renewing stem cell-like divisions of a restricted leading population that is highly proliferative. The fate of deposited cells, either within neuromasts or as interneuromast cells (in between deposited neuromasts) is not determined by any obvious stereotyped lineages. Instead, it is determined somewhat stochasitcailly, as a function of a cells distance from the center of a maturing protoneuromast. Together, our data provide a rigorous baseline for the behavior of the PLLp, which can be used to inform further study of this important model system.

Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 µm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

Instant super-resolution imaging in live cells and embryos via analog image processing.

Existing super-resolution fluorescence microscopes compromise acquisition speed to provide subdiffractive sample information. We report an analog implementation of structured illumination microscopy that enables three-dimensional (3D) super-resolution imaging with a lateral resolution of 145 nm and an axial resolution of 350 nm at acquisition speeds up to 100 Hz. By using optical instead of digital image-processing operations, we removed the need to capture, store and combine multiple camera exposures, increasing data acquisition rates 10- to 100-fold over other super-resolution microscopes and acquiring and displaying super-resolution images in real time. Low excitation intensities allow imaging over hundreds of 2D sections, and combined physical and computational sectioning allow similar depth penetration to spinning-disk confocal microscopy. We demonstrate the capability of our system by imaging fine, rapidly moving structures including motor-driven organelles in human lung fibroblasts and the cytoskeleton of flowing blood cells within developing zebrafish embryos.

Lef1 regulates Dusp6 to influence neuromast formation and spacing in the zebrafish posterior lateral line primordium.

The posterior lateral line primordium (PLLp) migrates caudally and periodically deposits neuromasts. Coupled, but mutually inhibitory, Wnt-FGF signaling systems regulate proto-neuromast formation in the PLLp: FGF ligands expressed in response to Wnt signaling activate FGF receptors and initiate proto-neuromast formation. FGF receptor signaling, in turn, inhibits Wnt signaling. However, mechanisms that determine periodic neuromast formation and deposition in the PLLp remain poorly understood. Previous studies showed that neuromasts are deposited closer together and the PLLp terminates prematurely in lef1-deficient zebrafish embryos. It was suggested that this results from reduced proliferation in the leading domain of the PLLp and/or premature incorporation of progenitors into proto-neuromasts. We found that rspo3 knockdown reduces proliferation in a manner similar to that seen in lef1 morphants. However, it does not cause closer neuromast deposition or premature termination of the PLLp, suggesting that such changes in lef1-deficient embryos are not linked to changes in proliferation. Instead, we suggest that they are related to the role of Lef1 in regulating the balance of Wnt and FGF functions in the PLLp. Lef1 determines expression of the FGF signaling inhibitor Dusp6 in leading cells and regulates incorporation of cells into neuromasts; reduction of Dusp6 in leading cells in lef1-deficient embryos allows new proto-neuromasts to form closer to the leading edge. This is associated with progressively slower PLLp migration, reduced spacing between deposited neuromasts and premature termination of the PLLp system.

Building the posterior lateral line system in zebrafish.

The posterior lateral line (pLL) in zebrafish has emerged as an excellent system to study how a sensory organ system develops. Here we review recent studies that illustrate how interactions between multiple signaling pathways coordinate cell fate,morphogenesis, and collective migration of cells in the posterior lateral line primordium. These studies also illustrate how the pLL system is contributing much more broadly to our understanding of mechanisms operating during the growth, regeneration, and self-organization of other organ systems during development and disease.