Research
- Evolution and development of diverse morphological phenotypes in domesticated ornamental fish
- Dynamic signaling during pattern formation (head formation and maintenance) in Hydra
- Entrainment and synchronization of signaling oscillations during mouse somitogenesis
- Directed evolution strategies for top-down engineering of complex microbial consortia
Evolution and development of diverse morphological phenotypes in domesticated ornamental fish
On the independent irritability of goldfish eggs and embryos – a living communication on the rhythmic yolk contractions in goldfish
Paul Gerald Layague Sanchez, Chen-Yi Wang, Ing-Jia Li, Kinya G. Ota
Abstract: Rhythms play an important role in the precise spatiotemporal regulation of biological processes during development and patterning of embryos. We here investigate the rhythmic contractions of the yolk during early development of the goldfish Carassius auratus. We quantify these contractions and record robust and persistent rhythmic yolk movements that are not seen in closely-related species (carp and zebrafish). We report that yolk contractions are an intrinsic emergent property of the egg, i.e. goldfish eggs are independently irritable / excitable. These contractions do not require sperm entry / fertilization nor cell division, and they notably emerge at a precise time — suggesting that goldfish eggs are able to measure elapsed time from what we infer to be egg activation. As the yolk itself is known to confer critical cues for early dorsoventral (DV) patterning of teleost embryos, we hypothesize that its contractions in goldfish may influence the patterning process of this species. Indeed, we find that embryos in conditions that result in ventralized phenotypes (i.e. goldfish embryos acutely treated with microtubule-depolymerizing drug nocodazole and embryos of the twin-tail goldfish strain Oranda) display altered yolk contraction dynamics (i.e. faster and/or stronger contractions). We aim to uncover whether the yolk contractions happening during early development of domesticated goldfish are the licensing process which explain the variety of novel DV patterning phenotypes naturally-observed in this species (e.g. twin-tail and dorsal-finless strains) and which are instead not found among closely-related species (e.g. carp) whose yolks do not contract.Evolutionary insights into muscle fiber distribution in the twin tails of ornamental goldfish
Kinya Ota, Gembu Abe, Chen-Yi Wang, Ing-Jia Li, Paul Gerald Layague Sanchez, Tzu-Chin Chi
Abstract: Twin-tail ornamental goldfish have a bifurcated caudal fin with a morphology that is extremely diverged from the conventional body plan of the vertebrates. Here, we investigate the musculoskeletal histology of this bifurcated caudal fin. From some of the investigated twin-tail goldfish, we found a twin-tail goldfish specific muscle (hereafter referred to as the "medial caudal muscle") between left and right bifurcated caudal fin skeletons. Our immunohistochemical analyses revealed that the medial caudal muscle showed mediolaterally biased distribution patterns of the slow and fast muscle fibers. Similar distribution patterns were also commonly observed in several deep muscles of wild-type goldfish as well as zebrafish, suggesting that these fishes share the same molecular developmental mechanisms even though their morphologies are highly diverged. These findings provide empirical evidence to consider how the histological features of a newly emerged morphology are influenced by pre-existing highly conserved developmental mechanisms.Dynamic signaling during pattern formation (head formation and maintenance) in Hydra
The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra
Matthias Christian Vogg, Jaroslav Ferenc, Wanda Christa Buzgariu, Chrystelle Perruchoud, Paul Gerald Layague Sanchez, Leonardo Beccari, Clara Nuninger, Youn Le Cras, Céline Delucinge-Vivier, Panagiotis Papasaikas, Stéphane Vincent, Brigitte Galliot, Charisios D Tsiairis
Abstract: The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.Related links:
The Wnt/β-catenin/TCF/Sp5/Zic4 gene network that regulates head organizer activity in Hydra is differentially regulated in epidermis and gastrodermis
Laura Iglesias Ollé, Chrystelle Perruchoud, Paul Gerald Layague Sanchez, Matthias Christian Vogg, Brigitte Galliot
Abstract: In Hydra, head formation depends on Wnt/β-catenin signaling, which positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in the epidermis or gastrodermis, we show that in intact animals, epidermal HySp5:GFP is expressed strongly apically and weakly along the body column, while gastrodermal HySp5:GFP is also maximally expressed apically but absent from the oral region, and remains high along the upper body column. During apical regeneration, gastrodermal HySp5:GFP appears early and diffusely, epidermal HySp5:GFP later. Upon alsterpaullone treatment, apical HySp5:GFP expression is shifted to the body column where epidermal HySp5:GFP transiently forms ectopic circular figures. After β-catenin(RNAi), only epidermal HySp5:GFP is down-regulated, while pseudo-bud structures expressing gastrodermal HySp5:GFP develop. Sp5(RNAi) highlights the negative autoregulation of Sp5 in epidermis, involving direct binding of Sp5 to its own promoter as observed in human HEK293T cells. In these cells, HyZic4, which can interact with huTCF1, regulates Wnt3 negatively and Sp5 positively. This differential regulation of the Wnt/β-catenin/TCF/Sp5/Zic4 network in epidermis and gastrodermis highlights distinct architectures and patterning roles in the hypostome, tentacle and body column, as well as distinct regulations in homeostatic and developmental organizers.Related link:
Entrainment and synchronization of signaling oscillations during mouse somitogenesis
Entrainment of coupled, phase-shifted signaling oscillations in the presomitic mesoderm
Paul Gerald Layague Sanchez
Summary: Synchronization is everywhere in nature. It is an emergent property arising in systems of interacting oscillatory entities, regardless if these entities are physical (e.g. in an electrical circuit) or biological (e.g. a group of fireflies). In vertebrate embryos, synchronization of intracellular signaling oscillations regulates the precise and periodic formation of somites, the precursors of vertebrae. In this system, oscillations are coordinated between neighbors via intercellular coupling, and such coordination results in a phase shift between oscillations, giving the impression of a spatiotemporal wave pattern travelling through the presomitic mesoderm (PSM) from posterior to anterior. Where this wave arrests at the anterior PSM, a new somite forms. The timing of such an event is mediated by the segmentation clock, an elaborate molecular signaling network between Notch, Wnt, and FGF signaling.While there are numerous studies focusing on the molecular details underlying such spatiotemporal regulation from the bottom-up, research delving into (1) the nature and mechanism of its synchronization and (2) its impact on tissue patterning during embryogenesis remains limited. To address this, we thus instead focus on a principles-based, top-down, approach. Accordingly, we recently developed a microfluidics-based experimental platform allowing entrainment of the signaling oscillations in the PSM to periodic pulses of signaling modulators, leveraging fundamental entrainment principles that have also been studied in other complex physical and biological oscillatory systems (e.g. the circadian clock).
In this current research, we use such experimental platform to map Arnold tongues, to systematically control both the period and the phase of oscillations in the PSM, and to precisely modulate the segmentation clock. We report how the systems-level entrainment behavior of oscillations in an embryonic tissue follows dynamical systems theory, despite its complexity. Furthermore, we uncover- and elaborate on a peculiar behavior in our system (i.e. emergence of a period gradient even upon tissue-level entrainment), providing insight into the nature of the underlying oscillatory network in the PSM. This finding has enabled us to generate testable hypotheses about the importance of the period gradient for the processing of spatiotemporal cues and proper tissue patterning. Moreover, experiments with intact embryonic tissue have allowed us to link modulation of the segmentation clock and its consequences on patterning of the PSM. We specifically record observations in apparent contradiction with traditional interpretations of a well-known model of periodic patterning during somitogenesis (i.e. the classical clock and wavefront model). Our observations instead support the proposition that the oscillatory dynamics encode both the timing and spacing of somite formation.
More generally, we here highlight the power of our experimental approach to precisely control the period and phase of a complex spatially-extended system of coupled and phase-shifted oscillations in an embryonic tissue, which had not been possible before using genetics and pharmacological intervention. We hope this research provides further experimental evidence of the universality of fundamental entrainment principles, and offers an alternative top-down approach to the study of synchronization of biological oscillations in embryonic development.
Arnold tongue entrainment reveals dynamical principles of the embryonic segmentation clock
Paul Gerald Layague Sanchez, Victoria Mochulska, Christian Mauffette Denis, Gregor Mönke, Takehito Tomita, Nobuko Tsuchida-Straeten, Yvonne Petersen, Katharina F. Sonnen, Paul François, Alexander Aulehla
Abstract: Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here, we aim to understand a complex system, that is, segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.Related links:
Glycolysis–Wnt signaling axis tunes developmental timing of embryo segmentation
Hidenobu Miyazawa, Jona Rada, Paul Gerald Layague Sanchez, Emilia Esposito, Daria Bunina, Charles Girardot, Judith Zaugg, Alexander Aulehla
Abstract: The question of how metabolism impacts development is seeing a renaissance. How metabolism exerts instructive signaling functions is one of the central issues that need to be resolved. We tackled this question in the context of mouse embryonic axis segmentation. Previous studies have shown that changes in central carbon metabolism impact Wnt signaling and the period of the segmentation clock, which controls the timing of axis segmentation. Here, we reveal that glycolysis tunes the segmentation clock period in an anti-correlated manner: higher glycolytic flux slows down the clock, and vice versa. Transcriptome and gene regulatory network analyses identified Wnt signaling and specifically the transcription factor Tcf7l2, previously associated with increased risk for diabetes, as potential mechanisms underlying flux-dependent control of the clock period. Critically, we show that deletion of the Wnt antagonist Dkk1 rescued the slow segmentation clock phenotype caused by increased glycolysis, demonstrating that glycolysis instructs Wnt signaling to control the clock period. In addition, we demonstrate metabolic entrainment of the segmentation clock: periodic changes in the levels of glucose or glycolytic sentinel metabolite fructose 1,6-bisphosphate (FBP) synchronize signaling oscillations. Notably, periodic FBP pulses first entrained Wnt signaling oscillations and subsequently Notch signaling oscillations. We hence conclude that metabolic entrainment has an immediate, specific effect on Wnt signaling. Combined, our work identifies a glycolysis-FBP-Wnt signaling axis that tunes developmental timing, highlighting the instructive signaling role of metabolism in embryonic development.Related link: https://youtu.be/GnSpIhBxjnU
Directed evolution strategies for top-down engineering of complex microbial consortia
Engineering complex communities by directed evolution
Chang-Yu Chang, Jean C C Vila, Madeline Bender, Richard Li, Madeleine C Mankowski, Molly Bassette, Julia Borden, Stefan Golfier, Paul Gerald L Sanchez, Rachel Waymack, Xinwen Zhu, Juan Diaz-Colunga, Sylvie Estrela, Maria Rebolleda-Gomez, Alvaro Sanchez
Abstract: Directed evolution has been used for decades to engineer biological systems at or below the organismal level. Above the organismal level, a small number of studies have attempted to artificially select microbial ecosystems, with uneven and generally modest success. Our theoretical understanding of artificial ecosystem selection is limited, particularly for large assemblages of asexual organisms, and we know little about designing efficient methods to direct their evolution. Here, we have developed a flexible modelling framework that allows us to systematically probe any arbitrary selection strategy on any arbitrary set of communities and selected functions. By artificially selecting hundreds of in silico microbial metacommunities under identical conditions, we first show that the main breeding methods used to date, which do not necessarily let communities reach their ecological equilibrium, are outperformed by a simple screen of sufficiently mature communities. We then identify a range of alternative directed evolution strategies that, particularly when applied in combination, are well suited for the top-down engineering of large, diverse and stable microbial consortia. Our results emphasize that directed evolution allows an ecological structure–function landscape to be navigated in search of dynamically stable and ecologically resilient communities with desired quantitative attributes.Related links: