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Huisken, J.

Publications and source records attributed to Huisken, J..

At least 19 recordsLinked to original sources

Critical period plasticity enables credit assignment

Synaptic plasticity is often guided by instructive inputs to neural circuits, but learning only succeeds when these instructions reach neurons that mediate relevant outputs. This creates the credit assignment problem: how does a neuron receive instructions suited to its own behavioral function? Here we report a developmental mechanism that enables credit assignment by using instructive inputs to organize the downstream architecture through which learning is expressed. In the olivocerebellar learning system, the inferior olive provides instructive inputs that guide plasticity within the cerebellum. During circuit assembly in zebrafish, we find these same inputs regulate long-range cerebellar projections during a highly plastic, two-day critical period. Developmental experience caused specific maturation of projections to targets that were coactivated with olivary inputs. Mathematical theory and computational modeling show how the resulting architecture constrains later learning, such that a single input can sculpt downstream connectivity and then leverage that circuit to assign credit. Simulated learning became paradoxically more robust if we reduced plasticity after a developmental critical period, protecting key architecture from corruption during learning. Thus, instructive inputs can first build the circuits they later teach, coordinating development and learning to enable effective credit assignment.

neuroscience↗

Multichannel optical cochlear implants enable spectrally distinct auditory activity

When hearing fails, cochlear implants (CIs) restore auditory perception. Yet, coding of spectral information remains a bottleneck because each electrode broadly activates the auditory nerve. As light can be more conveniently confined, optical (o)CIs present an alternative. Here, we combined expression of the potent channelrhodopsin ChReef in spiral ganglion neurons (SGNs) with oCIs comprising 5-10 green LEDs in gerbils. This combination enabled systematic comparison of encoding intensity and spectral information by individual oCI channels to acoustic and electrical stimulation within a translationally feasible energy range. Recordings from the inferior colliculus (ICC) showed that ChReef aligned SGN light sensitivity with LED radiant fluxes: ICC activity had thresholds <200 nJ and reached a maximum equivalent to that achieved with 51 dB SPL pure tones. Multichannel oCIs enabled tonotopically ordered, spectrally distinct stimulation that more closely resembled acoustic stimulation than did electrical stimulation. Some LEDs elicited multiple spectral peaks at higher intensities. Linear discriminant analysis of ICC activity indicated improved channel discriminability for optical over electrical stimulation. In summary, ,micro-joule oCI-stimulation achieves improved spectral resolution over state-of-the-art electrical stimulation.

neuroscience↗

CochleaNet: deep learning-based image analysis for cochlear connectomics and gene therapy

With the emergence of gene and optogenetic therapies targeting deafness, the comprehensive analysis of the molecular anatomy and physiology of the cochlea has become ever more important. Here, we introduce CochleaNet, a deep learning-based framework to analyze volumetric imaging data obtained by light-sheet microscopy of decalcified, cleared and fluorescently labeled cochleae. CochleaNet covers the workflow from reconstruction of the cochlea to segmentation of inner hair cells, spiral ganglion neurons and their afferent synapses, to analyzing the expression of gene therapy products. We validated CochleaNet by comparison to manual image analysis. Trained on high isotropic resolution mouse data, CochleaNet was also applicable to the cochlea of the gerbil, another relevant animal model, and lower-resolution mouse data from a commercially available microscope. We conclude that the combination of light-sheet microscopy and image analysis with CochleaNet paves the way for rapid and reliable quantification of cochlear molecular anatomy and preclinical gene therapy outcomes.

neuroscience↗

Leonardo: a toolset to correct sample-induced artifacts in light sheet microscopy images

Selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) is the method of choice for studying morphogenesis and function in biological specimens over extended periods, as it permits gentle and rapid volumetric imaging. In inhomogeneous samples, however, sample-induced artifacts, including light absorption, scattering, and refraction, can impact the image quality, particularly as the focal plane gets deeper into the sample. Here, we present Leonardo, the first toolbox designed to address the major sample-induced artifacts by using two modules: (1) DeStripe removes stripe artifacts in SPIM caused by light absorption while preserving fine sample structures; (2) Fuse reconstructs a single high-quality image from dualsided illumination and/or dual-sided detection, while eliminating blur and optical distortions caused by light scattering and refraction. The efficacy of Leonardo is validated on a wide range of biological samples, from minimally invasive experiments on sensitive specimens (translucent embryonic and optically opaque larval zebrafish) to cleared mouse samples up to two centimeters in size. We provide model code and a Napari-based graphical user interface, enabling the SPIM community to easily apply Leonardo to advance light sheet imaging of inhomogeneous and complex specimens.

bioinformatics↗

Isotropic, aberration-corrected light sheet microscopy for rapid high-resolution imaging of cleared tissue

Light sheet microscopy is the ideal technique for multiscale imaging of large and cleared tissues, and it is desirable to achieve the highest possible isotropic resolution across the entire sample. However, isotropic resolution for a centimeter-sized sample has only been achieved with slow and often aberrated, axially scanned light sheets, resulting in a low resolution of several micrometers. Here, we introduce a compact, high-speed light sheet fluorescence microscope with isotropic sub-micron resolution optimized for cleared tissue. We introduce three major opto-mechanical innovations using off-the-shelf optics to achieve an isotropic resolution of 850 nm across samples up to 1 cm3 and refractive indices ranging from 1.33 to 1.56, using mechanical tiling with a field of view of 800 {micro}m x 800 {micro}m. We show that combining an air objective and a meniscus lens achieves an axially swept light sheet with sub-micron diffraction-limited resolution and aberration correction. The effective field of view is increased 2-fold by correcting the field curvature of the light sheet with a concave mirror in the remote focusing unit. Furthermore, the imaging speed is enhanced 10-fold by adapting the light sheets motion with a closed-loop feedback, reaching 100 frames per second while maintaining isotropic resolution across the large field of view. Finally, we showcase the performance of our light sheet system for imaging from subcellular up to centimeter scale in cleared zebrafish, mouse cochlea, and mouse brain using various clearing methods.

developmental biology↗

In sickness and in health: RNA-Seq finds viruses associated with mutualist quality in the Amazonian plant-ant Allomerus octoarticulatus

Ant-plant symbioses are classic examples of mutualism in which ant "bodyguards" defend myrmecophytic plants against enemies in exchange for nest sites and often food. We used RNA-Seq to profile the transcriptomes of Allomerus octoarticulatus ant workers, which aggressively defend the Amazonian plant Cordia nodosa against herbivores, but to varying degrees. Field behavioral assays with herbivores in the Peruvian Amazon showed striking variation among colonies in the relative zeal with which A. octoarticulatus workers defend their host plant. Highly effective and ineffective bodyguards differed in their gene expression profiles, which revealed viral infections significantly associated with ant bodyguarding behavior. Transcripts from eight new positive-sense single-stranded RNA viruses were differentially expressed between colonies with high- or low-quality bodyguards. Colonies of high- and low-quality bodyguards were infected by distinct viruses, including viruses clustering phylogenetically with viruses known to cause aggression or reduced locomotion, respectively, in bees. Gene expression, including of immunity-related genes, also differed between broodcare workers and bodyguard ants, suggesting bodyguarding is a distinct worker task. Ant colony health and viral infections may influence ant cooperation with plants in ant-plant mutualisms. Significance statementMany animals have evolved to cooperate with plants in plant-animal mutualisms, including in pollination, seed dispersal, and defensive ant-plant interactions. Although the ecology of these interactions is well studied, little is known about the molecular mechanisms involved in animal cooperation with plants. Studying an Amazonian ant-plant symbiosis, we combined field assays of ant behavior with RNA-Seq to profile the transcriptomes of ants that vary in their quality as defensive plant "bodyguards." Bodyguard ants that effectively defended plants against herbivores expressed different genes than both ineffective bodyguards and broodcare workers. Surprisingly, many genes differentially expressed between high- and low-quality bodyguards were from viruses, and not ants. Thus, we linked viral infections to mutualist quality in this ant-plant symbiosis.

ecology↗

T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository

Selecting and implementing a tissue-clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue-clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that many articles do not provide sufficient detail to replicate or reproduce experimental results. To help address this issue, we have developed a novel, freely available repository of tissue-clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue-clearing protocols did not perform well (negative results). The goal of T-CLEARE is to provide a forum for the community to share evaluations and modifications of tissue-clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.

bioinformatics↗

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

bioengineering↗

Image restoration of degraded time-lapse microscopy data mediated by infrared-imaging.

Time-lapse fluorescence microscopy is key to unraveling the processes underpinning biological development and function. However, living systems, by their nature, permit only a limited toolbox for interrogation. Consequently, following time-lapses, expended samples contain untapped information that is typically discarded. Herein we employ convolutional neural networks (CNNs) to augment the live imaging data using this complementary information. In particular, live, deep tissue imaging is limited by the spectral range of live-cell compatible probes/fluorescent proteins. We demonstrate that CNNs may be used to restore deep-tissue contrast in GFP-based time-lapse imaging using paired final-state datasets acquired using infrared dyes and improve information content accordingly. Surprisingly, the networks are remarkably robust over a wide range of developmental times. We employ said network to GFP time-lapse images captured during zebrafish and drosophila embryo/larval development and demonstrate live, deep tissue image contrast.

developmental biology↗

A versatile pattern-forming cortical circuit based on Rho, F-actin, Ect2 and RGA-3/4

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner and, when co-expressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces a range of cortical behaviors from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho and F-actin form the core of a circuit that drives a diverse range of cortical behaviors, and demonstrate that the immature oocyte is a powerful model for characterizing these dynamics. SummaryMichaud et al. identify Ect2 and RGA-3/4 as core components of the cortical excitability circuit associated with cytokinesis. Additionally, they demonstrate that the immature Xenopus oocyte is a powerful model for characterizing excitable dynamics.

cell biology↗

KLC4 shapes axon arbors during development and mediates adult behavior

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.

neuroscience↗

Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

microbiology↗

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2-expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.

developmental biology↗

linus: Conveniently explore, share and present large-scale biological trajectory data from a web browser.

In biology, we are often confronted with information-rich, large-scale trajectory data, but exploring and communicating patterns in such data is often a cumbersome task. Ideally, the data should be wrapped with an interactive visualisation in one concise package that makes it straightforward to create and test hypotheses collaboratively. To address these challenges, we have developed a tool, linus, which makes the process of exploring and sharing 3D trajectories as easy as browsing a website. We provide a python script that reads trajectory data and enriches them with additional features, such as edge bundling or custom axes and generates an interactive web-based visualisation that can be shared offline and online. The goal of linus is to facilitate the collaborative discovery of patterns in complex trajectory data.

bioinformatics↗

In vivo lineage conversion of vertebrate muscle into early endoderm-like cells

The extent to which differentiated cells, while remaining in their native microenvironment, can be reprogrammed to assume a different identity will reveal fundamental insight into cellular plasticity and impact regenerative medicine. To investigate in vivo cell lineage potential, we leveraged the zebrafish as a practical vertebrate platform to determine factors and mechanisms necessary to induce differentiated cells of one germ layer to adopt the lineage of another. We discovered that ectopic co-expression of Sox32 and Oct4 in several non-endoderm lineages, including skeletal muscle, can specifically trigger an early endoderm genetic program in a cell-autonomous manner. Gene expression, live imaging, and functional studies reveal that the endoderm-induced muscle cells lose muscle gene expression and morphology, while specifically gaining endoderm organogenesis markers, such as the pancreatic specification genes, hhex and ptf1a, via a mechanism resembling normal development. Endoderm induction by a pluripotent defective form of Oct4, endoderm markers appearing prior to loss of muscle cell morphology, a lack of dependence on cell division, and a lack of mesoderm, ectoderm, dedifferentiation, and pluripotency gene activation, together, suggests that reprogramming is endoderm specific and occurs via direct lineage conversion. Our work demonstrates that within a vertebrate animal, stably differentiated cells can be induced to directly adopt the identity of a completely unrelated cell lineage, while remaining in a distinct microenvironment, suggesting that differentiated cells in vivo may be more amenable to lineage conversion than previously appreciated. This discovery of possibly unlimited lineage potential of differentiated cells in vivo challenges our understanding of cell lineage restriction and may pave the way towards a vast new in vivo supply of replacement cells for degenerative diseases such as diabetes.

developmental biology↗

Multi-sample SPIM image acquisition, processing and analysis of vascular growth in zebrafish

To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously and automatically process and analyze the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing, and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented with a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature, and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies.\n\nSummary statementWe present a dedicated multi-sample light sheet imaging, processing and analysis platform and demonstrate its value for studies of vascular growth in zebrafish.

developmental biology↗

Basal extrusion drives cell invasion and mechanical stripping of E-cadherin

Metastasis is the predominant reason that patients succumb to cancer, yet the mechanisms that drive initial tumor cell invasion are poorly understood. We previously discovered that crowding-induced apical extrusion drives most epithelial cell death, critical to maintaining constant cell densities. Oncogenic mutations can disrupt apical cell extrusion, instead causing masses to form and aberrant basal extrusion. Using transparent zebrafish epidermis to model simple epithelia, we can image invasion events live at high resolution. We find that KRas/p53-transformed cells form masses and, at completely independent sites, invade by basal extrusion. Basal extrusion also causes invading cells to simultaneously mechanically shed their entire apical membranes and E-cadherin. Once cells invade the underlying tissue, they migrate throughout the body, divide, enter the bloodstream, and become different cell types. KRas-transformation makes cells intrinsically invasive by increasing basal extrusion rates; collaborating mutations in p53 allow disseminated cells to survive at distant sites.

cancer biology↗

Endothelial cells form transient Notch-dependent NO-containing cystic structures during zebrafish cerebrovascular development

Endothelial cell behaviour during blood vessel formation is highly complex and dynamic. Transgenic zebrafish have provided many new insights into these processes, due to their ability to provide detailed in vivo imaging.\n\nWe here report a previously undescribed endothelial cell behaviour during zebrafish embryonic development. Endothelial cells of the cerebral vessels of 3-5d post fertilisation embryos extruded large membranous spherical structures. These were only found on the cerebral vessels, and did not detach from the parent vessel, instead regressing back into the endothelial cell. These structures did not communicate with the vessel lumen, exhibited periodic oscillations in size and shape, and were enriched with filamentous actin at their neck. Due to their unknown nature and spherical appearance we termed these structures kugeln (German for sphere).\n\nPharmacological inhibition of vascular endothelial growth factor (VEGF) signalling significantly increased kugel number while Notch inhibition significantly reduced both kugel number and diameter. Kugeln contain little cytoplasm, but are highly positive for nitric oxide (NO) reactivity, suggesting they represent a novel NO containing organelle specific to the cerebral vessels.

developmental biology↗