Generation of patterns in the paraxial mesoderm.

The Segmentation Clock is a tissue-level patterning system that enables the segmentation of the vertebral column precursors into transient multicellular blocks called somites. This patterning system comprises a set of elements that are essential for correct segmentation. Under the so-called "Clock and Wavefront" model, the system consists of two elements, a genetic oscillator that manifests itself as traveling waves of gene expression, and a regressing wavefront that transforms the temporally periodic signal encoded in the oscillations into a permanent spatially periodic pattern of somite boundaries. Over the last twenty years, every new discovery about the Segmentation Clock has been tightly linked to the nomenclature of the "Clock and Wavefront" model. This constrained allocation of discoveries into these two elements has generated long-standing debates in the field as what defines molecularly the wavefront and how and where the interaction between the two elements establishes the future somite boundaries. In this review, we propose an expansion of the "Clock and Wavefront" model into three elements, "Clock", "Wavefront" and signaling gradients. We first provide a detailed description of the components and regulatory mechanisms of each element, and we then examine how the spatiotemporal integration of the three elements leads to the establishment of the presumptive somite boundaries. To be as exhaustive as possible, we focus on the Segmentation Clock in zebrafish. Furthermore, we show how this three-element expansion of the model provides a better understanding of the somite formation process and we emphasize where our current understanding of this patterning system remains obscure.

Open-source microscope add-on for structured illumination microscopy.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

Single-cell transcriptional dynamics in a living vertebrate.

The ability to quantify transcriptional dynamics in individual cells via live imaging has revolutionized our understanding of gene regulation. However, such measurements are lacking in the context of vertebrate embryos. We addressed this deficit by applying MS2-MCP mRNA labeling to the quantification of transcription in zebrafish, a model vertebrate. We developed a platform of transgenic organisms, light sheet fluorescence microscopy, and optimized image analysis that enables visualization and quantification of MS2 reporters. We used these tools to obtain the first single-cell, real-time measurements of transcriptional dynamics of the segmentation clock. Our measurements challenge the traditional view of smooth clock oscillations and instead suggest a model of discrete transcriptional bursts that are organized in space and time. Together, these results highlight how measuring single-cell transcriptional activity can reveal unexpected features of gene regulation and how this data can fuel the dialogue between theory and experiment.

Identification of side effects of COVID-19 drug candidates on embryogenesis using an integrated zebrafish screening platform.

Drug repurposing is an important strategy in COVID-19 treatment, but many clinically approved compounds have not been extensively studied in the context of embryogenesis, thus limiting their administration during pregnancy. Here we used the zebrafish embryo model organism to test the effects of 162 marketed drugs on cardiovascular development. Among the compounds used in the clinic for COVD-19 treatment, we found that Remdesivir led to reduced body size and heart functionality at clinically relevant doses. Ritonavir and Baricitinib showed reduced heart functionality and Molnupiravir and Baricitinib showed effects on embryo activity. Sabizabulin was highly toxic at concentrations only 5 times higher than Cmax and led to a mean mortality of 20% at Cmax. Furthermore, we tested if zebrafish could be used as a model to study inflammatory response in response to spike protein treatment and found that Remdesivir, Ritonavir, Molnupiravir, Baricitinib as well as Sabizabulin counteracted the inflammatory response related gene expression upon SARS-CoV-2 spike protein treatment. Our results show that the zebrafish allows to study immune-modulating properties of COVID-19 compounds and highlights the need to rule out secondary defects of compound treatment on embryogenesis. All results are available on a user friendly web-interface https://share.streamlit.io/alernst/covasc_dataapp/main/CoVasc_DataApp.py that provides a comprehensive overview of all observed phenotypic effects and allows personalized search on specific compounds or group of compounds. Furthermore, the presented platform can be expanded for rapid detection of developmental side effects of new compounds for treatment of COVID-19 and further viral infectious diseases.

A retrospective study of canine oral extramedullary plasmacytoma over a 15-year period (July 2004-July 2019): Treatment, histologic parameters and clinical outcomes.

A total of 45 cases of canine oral extramedullary plasmacytomas (EMPs) presented to a tertiary referral institution over a 15-year period were examined. Histologic sections of 33 of these cases were examined for histopathologic prognostic indicators. Patients underwent variable treatment including surgical intervention, chemotherapy and/or radiation therapy. Long term survival was observed in the majority of dogs with a median survival time of 973 days (2-4315 days). However, almost 1/3 of dogs had progression of plasma cell disease, including two cases with myeloma-like progression. Histologic characterization of these tumours did not reveal criteria to predict tumour malignancy. However, cases without tumour progression did not exceed 28 mitotic figures in ten 400× fields (2.37 mm2 ). All cases with tumour related death showed at least moderate nuclear atypia. Oral EMPs may represent a local manifestation of systemic plasma cell disease or singular focal neoplasia.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Deconstructing body axis morphogenesis in zebrafish embryos using robot-assisted tissue micromanipulation.

Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations.

Preparation of Single-Somite Explants from Zebrafish Embryos.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

Left-right symmetry of zebrafish embryos requires somite surface tension.

The body axis of vertebrate embryos is periodically segmented into bilaterally symmetric pairs of somites1,2. The anteroposterior length of somites, their position and left-right symmetry are thought to be molecularly determined before somite morphogenesis3,4. Here we show that, in zebrafish embryos, initial somite anteroposterior lengths and positions are imprecise and, consequently, many somite pairs form left-right asymmetrically. Notably, these imprecisions are not left unchecked and we find that anteroposterior lengths adjust within an hour after somite formation, thereby increasing morphological symmetry. We find that anteroposterior length adjustments result entirely from changes in somite shape without change in somite volume, with changes in anteroposterior length being compensated by corresponding changes in mediolateral length. The anteroposterior adjustment mechanism is facilitated by somite surface tension, which we show by comparing in vivo experiments and in vitro single-somite explant cultures using a mechanical model. Length adjustment is inhibited by perturbation of molecules involved in surface tension, such as integrin and fibronectin. By contrast, the adjustment mechanism is unaffected by perturbations to the segmentation clock, therefore revealing a distinct process that influences morphological segment lengths. We propose that tissue surface tension provides a general mechanism to adjust shapes and ensure precision and symmetry of tissues in developing embryos.

Oropharyngeal Shedding of Gammaherpesvirus DNA by Cats, and Natural Infection of Salivary Epithelium.

Felis catus gammaherpesvirus-1 (FcaGHV1), a novel candidate oncogenic virus, infects cats worldwide. Whether the oropharynx is a site of virus shedding and persistence, and whether oronasal carcinomas harbor FcaGHV1 nucleic acid were investigated. In a prospective molecular epidemiological study, FcaGHV1 DNA was detected by cPCR in oropharyngeal swabs from 26/155 (16.8%) of cats. Oropharyngeal shedding was less frequently detected in kittens ≤3 months of age (5/94, 5.3%) than in older animals; >3 months to ≤1 year: 8/26, 30.8%, (p = 0.001, OR 7.91, 95% CI (2.320, 26.979)); >1 year to ≤6 years: 10/20, 50%, (p < 0.001, OR 17.8 95% CI (5.065, 62.557)); >6 years: 3/15, 33% (p = 0.078). Provenance (shelter-owned/privately owned) was not associated with shedding. In situ hybridization (ISH) identified FcaGHV1-infected cells in salivary glandular epithelium but not in other oronasal tissues from two of three cats shedding viral DNA in the oropharynx. In a retrospective dataset of 11 oronasopharyngeal carcinomas, a single tumor tested positive for FcaGHV1 DNA by ISH, a papillary carcinoma, where scattered neoplastic cells showed discrete nuclear hybridization. These data support the oronasopharynx as a site of FcaGHV1 shedding, particularly after maternal antibodies are expected to decline. The salivary epithelium is identified as a potential site of FcaGHV1 persistence. No evidence supporting a role for FcaGHV1 in feline oronasal carcinomas was found in the examined tumours.

Towards a physical understanding of developmental patterning.

The temporal coordination of events at cellular and tissue scales is essential for the proper development of organisms, and involves cell-intrinsic processes that can be coupled by local cellular signalling and instructed by global signalling, thereby creating spatial patterns of cellular states that change over time. The timing and structure of these patterns determine how an organism develops. Traditional developmental genetic methods have revealed the complex molecular circuits regulating these processes but are limited in their ability to predict and understand the emergent spatio-temporal dynamics. Increasingly, approaches from physics are now being used to help capture the dynamics of the system by providing simplified, generic descriptions. Combined with advances in imaging and computational power, such approaches aim to provide insight into timing and patterning in developing systems.

From local resynchronization to global pattern recovery in the zebrafish segmentation clock.

Integrity of rhythmic spatial gene expression patterns in the vertebrate segmentation clock requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition causes defective segment boundaries. Whether deformation of the oscillating tissue complements local synchronization during patterning and segment formation is not understood. We combine theory and experiment to investigate this question in the zebrafish segmentation clock. We remove a Notch inhibitor, allowing resynchronization, and analyze embryonic segment recovery. We observe unexpected intermingling of normal and defective segments, and capture this with a new model combining coupled oscillators and tissue mechanics. Intermingled segments are explained in the theory by advection of persistent phase vortices of oscillators. Experimentally observed changes in recovery patterns are predicted in the theory by temporal changes in tissue length and cell advection pattern. Thus, segmental pattern recovery occurs at two length and time scales: rapid local synchronization between neighboring cells, and the slower transport of the resulting patterns across the tissue through morphogenesis.

Waiting on the Fringe: cell autonomy and signaling delays in segmentation clocks.

The rhythmic and sequential segmentation of the vertebrate body axis into somites during embryogenesis is governed by a multicellular, oscillatory patterning system called the segmentation clock. Despite many overt similarities between vertebrates, differences in genetic and dynamic regulation have been reported, raising intriguing questions about the evolution and conservation of this fundamental patterning process. Recent studies have brought insights into two important and related issues: (1) whether individual cells of segmentation clocks are autonomous oscillators or require cell-cell communication for their rhythm; and (2) the role of delays in the cell-cell communication that synchronizes the population of genetic oscillators. Although molecular details differ between species, conservation may exist at the level of the dynamics, hinting at rules for evolutionary trajectories in the system.

What are you synching about? Emerging complexity of Notch signaling in the segmentation clock.

The Segmentation clock is a population of cellular genetic oscillators, located in the posterior of the elongating vertebrate embryo, that governs the rhythmic and sequential segmentation of the body axis into somites. Somites are blocks of cells that give rise to the segmented anatomy of the adult, including the backbone, muscles and skin. Malfunction of the segmentation clock results in malformations of these structures, a condition termed congenital scoliosis in the clinic. In all vertebrates, the oscillating cells of the segmentation clock are coordinated in a wave pattern, such that each new wave corresponds to a new segment. Maintenance of this wave pattern is important for precise segmentation and requires the local synchronization of the cellular oscillators. Existing models of the segmentation clock have explored the role of the Delta-Notch intercellular signaling pathway primarily as a coupling mechanism between neighboring autonomous oscillators. Recent work challenges several aspects of this simplification, suggesting that the mechanism of synchronization is more complex and may differ between species, and that Notch signaling may do more than synchronize cells. Here, we first examine evidence and models concerning the role of Notch signaling in driving, maintaining and synchronizing the mouse clock, highlighting results emerging from ex vivo culture systems of mouse segmentation clock cells. We then compare this to synchronization in the zebrafish, where accumulating evidence suggests that Notch signaling impacts the amplitude of the oscillating signal, and discuss whether the amplitude itself is meaningful for segmentation. Finally, we review work showing that multiple Delta ligands are active in segmentation, and consider how an interplay between these ligands could confer effective Notch functions in the segmentation clock. These lines of enquiry suggest that synchronization and Notch signaling are more complex than previously described, and reveal exciting new avenues for investigation into the coordination and precision of patterning the early embryo.

Embryonic lateral inhibition as optical modes: An analytical framework for mesoscopic pattern formation.

Cellular checkerboard patterns are observed at many stages of embryonic development. We study an analytically tractable model for lateral inhibition and show that the steady states are analogous to optical phonons at the Γ point, which have the wave number k=0. We study the cases of cells arranged in linear and hexagonal lattices. To determine how the final pattern is selected it is necessary to take into account the granularity of the pattern and, analogously to solid-state physics, to redefine the basis and lattice sites in terms of a periodic crystal. The sites and basis are determined by looking at the symmetries of inhibitory interactions between cells. The redefined basis for cells placed in a linear lattice is composed by two cells which are embedded in another linear lattice, while for cells placed in a hexagonal lattice the redefined basis consists of three cells embedded in another hexagonal lattice. The pattern in hexagonal lattices can be driven into three different states: two of those states are periodic checkerboards and a third in which both periodic states coexist. These observations provides new predictions for experiments.

Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos.

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.

Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock.

Segmentation of the axial skeleton in amniotes depends on the segmentation clock, which patterns the paraxial mesoderm and the sclerotome. While the segmentation clock clearly operates in teleosts, the role of the sclerotome in establishing the axial skeleton is unclear. We severely disrupt zebrafish paraxial segmentation, yet observe a largely normal segmentation process of the chordacentra. We demonstrate that axial entpd5+ notochord sheath cells are responsible for chordacentrum mineralization, and serve as a marker for axial segmentation. While autonomous within the notochord sheath, entpd5 expression and centrum formation show some plasticity and can respond to myotome pattern. These observations reveal for the first time the dynamics of notochord segmentation in a teleost, and are consistent with an autonomous patterning mechanism that is influenced, but not determined by adjacent paraxial mesoderm. This behavior is not consistent with a clock-type mechanism in the notochord.

Small molecule screen in embryonic zebrafish using modular variations to target segmentation.

Small molecule in vivo phenotypic screening is used to identify drugs or biological activities by directly assessing effects in intact organisms. However, current screening designs may not exploit the full potential of chemical libraries due to false negatives. Here, we demonstrate a modular small molecule screen in embryonic zebrafish that varies concentration, genotype and timing to target segmentation disorders, birth defects that affect the spinal column. By testing each small molecule in multiple interrelated ways, this screen recovers compounds that a standard screening design would have missed, increasing the hit frequency from the chemical library three-fold. We identify molecular pathways and segmentation phenotypes, which we share in an open-access annotated database. These hits provide insight into human vertebral segmentation disorders and myopathies. This modular screening strategy is applicable to other developmental questions and disease models, highlighting the power of relatively small chemical libraries to accelerate gene discovery and disease study.

A framework for quantification and physical modeling of cell mixing applied to oscillator synchronization in vertebrate somitogenesis.

In development and disease, cells move as they exchange signals. One example is found in vertebrate development, during which the timing of segment formation is set by a 'segmentation clock', in which oscillating gene expression is synchronized across a population of cells by Delta-Notch signaling. Delta-Notch signaling requires local cell-cell contact, but in the zebrafish embryonic tailbud, oscillating cells move rapidly, exchanging neighbors. Previous theoretical studies proposed that this relative movement or cell mixing might alter signaling and thereby enhance synchronization. However, it remains unclear whether the mixing timescale in the tissue is in the right range for this effect, because a framework to reliably measure the mixing timescale and compare it with signaling timescale is lacking. Here, we develop such a framework using a quantitative description of cell mixing without the need for an external reference frame and constructing a physical model of cell movement based on the data. Numerical simulations show that mixing with experimentally observed statistics enhances synchronization of coupled phase oscillators, suggesting that mixing in the tailbud is fast enough to affect the coherence of rhythmic gene expression. Our approach will find general application in analyzing the relative movements of communicating cells during development and disease.

The Sweetness of Embryonic Elongation and Differentiation.

Metabolic pathways play a vital yet poorly understood role in embryogenesis. In this issue of Developmental Cell, Bulusu et al. (2017) and Oginuma et al. (2017) provide insights into the intricate relationship between metabolism and morphogenesis, showing that glycolysis facilitates body elongation and balances neural and mesodermal differentiation.