Tetrahedral serial multiview microscopy and image fusion for improved resolution and extent in stained zebrafish embryos.

BACKGROUND: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware. RESULTS: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be created from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent. CONCLUSION: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.

Size control of the inner ear via hydraulic feedback.

Animals make organs of precise size, shape, and symmetry but how developing embryos do this is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the developing inner ear. We find that fluid accumulation creates hydrostatic pressure in the lumen leading to stress in the epithelium and expansion of the otic vesicle. Pressure, in turn, inhibits fluid transport into the lumen. This negative feedback loop between pressure and transport allows the otic vesicle to change growth rate to control natural or experimentally-induced size variation. Spatiotemporal patterning of contractility modulates pressure-driven strain for regional tissue thinning. Our work connects molecular-driven mechanisms, such as osmotic pressure driven strain and actomyosin tension, to the regulation of tissue morphogenesis via hydraulic feedback to ensure robust control of organ size. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure.

The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Recovery of shape and size in a developing organ pair.

BACKGROUND: Paired organs in animals are largely bilaterally symmetric despite inherent noise in most biological processes. How is precise organ shape and size achieved during development despite this noise? Examining paired organ development is a challenge because it requires repeated quantification of two structures in parallel within living embryos. Here we combine bilateral quantification of morphology through time with asymmetric perturbations to study regulation of organ shape, size, and symmetry in developing organ pairs. RESULTS: We present quantitative live imaging tools to measure the shape and size of the developing inner ears on both the left and right side simultaneously over time. By quantifying variation between the left and right inner ear (intrinsic noise) and between different individuals (extrinsic noise), we find that initial variability decreases over time in normal development to achieve symmetry. Early asymmetry is increased by environmental stress, but symmetry is still recovered over subsequent developmental time. Using multiple unilateral perturbations including Fgf signaling and ultraviolet light, we find that growth can be adjusted to compensate for a range of initial size and shape differences. CONCLUSIONS: We propose that symmetry in developmental systems does not emerge through precise deterministic bilateral development, but rather through feedback mechanisms that adjust morphogenesis rates to account for variation. Developmental Dynamics 246:451-465, 2016. © 2017 Wiley Periodicals, Inc.

Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin.

Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.

Interplay of cell shape and division orientation promotes robust morphogenesis of developing epithelia.

Epithelial cells acquire functionally important shapes (e.g., squamous, cuboidal, columnar) during development. Here, we combine theory, quantitative imaging, and perturbations to analyze how tissue geometry, cell divisions, and mechanics interact to shape the presumptive enveloping layer (pre-EVL) on the zebrafish embryonic surface. We find that, under geometrical constraints, pre-EVL flattening is regulated by surface cell number changes following differentially oriented cell divisions. The division pattern is, in turn, determined by the cell shape distribution, which forms under geometrical constraints by cell-cell mechanical coupling. An integrated mathematical model of this shape-division feedback loop recapitulates empirical observations. Surprisingly, the model predicts that cell shape is robust to changes of tissue surface area, cell volume, and cell number, which we confirm in vivo. Further simulations and perturbations suggest the parameter linking cell shape and division orientation contributes to epithelial diversity. Together, our work identifies an evolvable design logic that enables robust cell-level regulation of tissue-level development.

Specified neural progenitors sort to form sharp domains after noisy Shh signaling.

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

ACME: automated cell morphology extractor for comprehensive reconstruction of cell membranes.

The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME).

Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing.

Forward genetic screens in zebrafish have identified >9000 mutants, many of which are potential disease models. Most mutants remain molecularly uncharacterized because of the high cost, time and labor investment required for positional cloning. These costs limit the benefit of previous genetic screens and discourage future screens. Drastic improvements in DNA sequencing technology could dramatically improve the efficiency of positional cloning in zebrafish and other model organisms, but the best strategy for cloning by sequencing has yet to be established. Using four zebrafish inner ear mutants, we developed and compared two approaches for 'cloning by sequencing': one based on bulk segregant linkage (BSFseq) and one based on homozygosity mapping (HMFseq). Using BSFseq we discovered that mutations in lmx1b and jagged1b cause abnormal ear morphogenesis. With HMFseq we validated that the disruption of cdh23 abolishes the ear's sensory functions and identified a candidate lesion in lhfpl5a predicted to cause nonsyndromic deafness. The success of HMFseq shows that the high intrastrain polymorphism rate in zebrafish eliminates the need for time-consuming map crosses. Additionally, we analyzed diversity in zebrafish laboratory strains to find areas of elevated diversity and areas of fixed homozygosity, reinforcing recent findings that genome diversity is clustered. We present a database of >15 million sequence variants that provides much of this approach's power. In our four test cases, only a single candidate single nucleotide polymorphism (SNP) remained after subtracting all database SNPs from a mutant's critical region. The saturation of the common SNP database and our open source analysis pipeline MegaMapper will improve the pace at which the zebrafish community makes unique discoveries relevant to human health.

Intron length increases oscillatory periods of gene expression in animal cells.

Introns may affect gene expression by increasing the time required to transcribe the gene. One way for extended transcription times to affect the behavior of a gene expression program is through a negative feedback loop. Here, we show that a logically engineered negative feedback loop in animal cells produces expression pulses, which have a broad time distribution that increases with intron length. These results in combination with mathematical models provide insight into what may produce the intron-dependent pulse distributions. We conclude that the long production time required for large intron-containing genes is significant for the behavior of gene expression programs.

Intron delays and transcriptional timing during development.

The time taken to transcribe most metazoan genes is significant because of the substantial length of introns. Developmentally regulated gene networks, where timing and dynamic patterns of expression are critical, may be particularly sensitive to intron delays. We revisit and comment on a perspective last presented by Thummel 16 years ago: transcriptional delays may contribute to timing mechanisms during development. We discuss the presence of intron delays in genetic networks. We consider how delays can impact particular moments during development, which mechanistic attributes of transcription can influence them, how they can be modeled, and how they can be studied using recent technological advances as well as classical genetics.

Enhancement of cell type specificity by quantitative modulation of a chimeric ligand.

Evolution modulates the quantitative characteristics of protein interactions and often uses combinations of weak interactions to achieve a particular specificity. We addressed how quantitative optimization might be used in the design of multidomain proteins, using a chimera containing epidermal growth factor (EGF) as a cell targeting element and interferon-alpha-2a (IFNalpha-2a) to initiate signal transduction. We first connected EGF and IFNalpha-2a via a linker that allows both ligands to bind to their receptors on a cell surface and then incorporated a series of mutations into the IFNalpha-2a portion that progressively decrease both the on rate and the dissociation constant of the IFNalpha-2a-IFNalpha receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFNalpha receptor in HeLa, A431, and engineered Daudi cells depends on the presence of EGF receptor on the same cell. The mutant chimeric proteins also inhibited proliferation of IFNalpha-sensitive cells in an EGF receptor-dependent manner. These results provide insights into the quantitative requirements for specific binding to multisubunit receptors and illustrate the value of a quantitative approach in the design of synthetic-biological constructs.

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription.

Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3' end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3' ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3' processing factor, CSTF2, had biases toward 3' ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA-protein complexes.

Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells.

BACKGROUND: Transcription by RNA polymerase II is regulated at many steps including initiation, promoter release, elongation and termination. Accumulation of RNA polymerase II at particular locations across genes can be indicative of sites of regulation. RNA polymerase II is thought to accumulate at the promoter and at sites of co-transcriptional alternative splicing where the rate of RNA synthesis slows. RESULTS: To further understand transcriptional regulation at a global level, we determined the distribution of RNA polymerase II within regions of the human genome designated by the ENCODE project. Hypophosphorylated RNA polymerase II localizes almost exclusively to 5' ends of genes. On the other hand, localization of total RNA polymerase II reveals a variety of distinct landscapes across many genes with 74% of the observed enriched locations at exons. RNA polymerase II accumulates at many annotated constitutively spliced exons, but is biased for alternatively spliced exons. Finally, RNA polymerase II is also observed at locations not in gene regions. CONCLUSION: Localizing RNA polymerase II across many millions of base pairs in the human genome identifies novel sites of transcription and provides insights into the regulation of transcription elongation. These data indicate that RNA polymerase II accumulates most often at exons during transcription. Thus, a major factor of transcription elongation control in mammalian cells is the coordination of transcription and pre-mRNA processing to define exons.