Andrew Charles Oates

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

Erik Mailand, Andrew Charles Oates, Ece Ozelci, Matthias Rüegg, Mahmut Selman Sakar

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.

2022

Preparation of Single-Somite Explants from Zebrafish Embryos

Sundar Ram Naganathan, Andrew Charles Oates

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.

JOURNAL OF VISUALIZED EXPERIMENTS2022

Reaction wavefront theory of notochord segment patterning

Andrew Charles Oates

The vertebrate axis is segmented into repetitive structures, the vertebrae. In fish, these segmented structures are thought to form from the paraxial mesoderm and the adjacent notochord. Recent work revealed an autonomous patterning mechanism in the zebrafish notochord, with inputs from the segmented paraxial mesoderm. The notochord pattern is established in a sequential manner, progressing from anterior to posterior. Building on this previous work, here, we propose a reaction wavefront theory describing notochord patterning in zebrafish. The pattern is generated by an activator-inhibitor reaction-diffusion mechanism. Cues from the paraxial mesoderm are introduced as a profile of inhibitor sinks. Reactions are turned on by a wavefront that advances from anterior to posterior. We show that this reaction wavefront ensures that a pattern is formed sequentially, in register with the cues, despite the presence of fluctuations. We find that the velocity and shape of the reaction wavefront can modulate the prevalence of defective patterns. Normal patterning is supported in a wide range of sink profile wavelengths, while a minimum sink strength is required for the pattern to follow the cues. The theory predicts that distinct defect types occur for small or large wavelengths. Thus, the reaction wavefront theory provides a possible scenario for notochord patterning, with testable predictions that prompt future experiments.

FRONTIERS MEDIA SA2022

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

Andrew Charles Oates

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.

ELIFE SCIENCES PUBLICATIONS LTD2021

Theory of time delayed genetic oscillations with external noisy regulation

Jose Negrete Jr, Andrew Charles Oates, Laurel Ann Rohde

Video Abstract Video Abstract: Theory of time delayed genetic oscillations with external noisy regulation We present a general theory of noisy genetic oscillators with externally regulated production rate and multiplicative noise. The observables that characterize the genetic oscillator are discussed, and it is shown how their statistics depend on the externally regulated production rate. We show that these observables have generic features that are observed in two different experimental systems: the expression of the circadian clock genes in fibroblasts, and in the transient and oscillatory dynamics of the segmentation clock genes observed in cells disassociated from zebrafish embryos. Our work shows that genetic oscillations with diverse biological contexts can be understood in a common framework based on a delayed negative feedback system, and regulator dynamics.

IOP PUBLISHING LTD2021

Patterning and mechanics of somite boundaries in zebrafish embryos

Sundar Ram Naganathan, Andrew Charles Oates

The body axis of vertebrates is subdivided into repetitive compartments called somites, which give rise primarily to the segmented architecture of the musculoskeletal system in the adult body. Somites form in a sequential and rhythmic manner in embryos and a physical boundary separates each somite from the rest of the unsegmented tissue and adjoining somites. Precise positioning of somite boundaries and determination of boundary cell fate in a select group of cells is thought to be driven by gene expression patterns and morphogen gradients. This pre-patterning step is followed by a mechanical process involving actomyosin activation in boundary cells and formation of an extracellular matrix that results in morphological boundary formation. While genes involved in somite boundary formation have been identified, there are many open questions about the underlying pre-patterning dynamics and mechanics and how these processes are coupled to generate a morphological boundary. Here, focusing on segmentation of zebrafish embryos as a model, we review pre-patterning processes critical for boundary formation and how cytoskeletal activity drives tissue separation. Our outlook is that this system holds exciting new avenues for unearthing general principles of boundary formation in developing embryos.

2020

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

Andrew Charles Oates, Olivier François Venzin

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.

ACADEMIC PRESS INC ELSEVIER SCIENCE2020

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

Rachna Narayanan, Andrew Charles Oates

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.

BIO-PROTOCOL2019

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

Jose Negrete Jr, Andrew Charles Oates

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 Gamma 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.

2019