The Lifeact-EGFP Quail: A Powerful Avian Model for Live Imaging of Actin Dynamics During Embryonic Morphogenesis

Основні поняття

The generation of a transgenic Lifeact-EGFP quail line enables high-resolution live imaging of actin structures and dynamics during key morphogenetic processes in the developing avian embryo.

Анотація

The authors report the generation of a transgenic Lifeact-EGFP quail line to investigate actin organization and dynamics during embryonic morphogenesis in vivo. This avian model allows for the visualization of actin structures from the subcellular to the tissue level, facilitating the study of previously intractable processes.
Key highlights:

Live imaging of cardiac progenitor cells reveals filopodia-dependent migration and contact-mediated stabilization of filopodia.
Protrusions in the developing neural tube appear to assist in the zippering of the neural folds during closure.
Apical constriction in the neuroepithelium occurs through a pulsatile, ratchet-like mechanism involving dynamic changes in the apical actin network.
Supracellular actin cables and multicellular rosettes form in the neural plate, contributing to the anisotropic bending of the tissue during neural tube formation.
The authors demonstrate the utility of the Lifeact-EGFP transgenic quail as a powerful model system for live in vivo investigations of the actin cytoskeleton during embryonic morphogenesis.

Статистика

Cardiac progenitor cell filopodia are on average 9.1μm +/-0.5μm long and have an average persistence time of 389.1 s +/-22.9 s.
Filopodia that contact the surrounding tissues are significantly longer (11.2μm +/-0.7μm) and more persistent (523.6 s +/-34.5 s) than those that do not make contact (7.2μm +/-0.4μm and 276.0 s +/-20.5 s).
The rate of area change is faster during constriction phases than expansion phases in apically constricting neuroepithelium cells.
The intensity of Lifeact-EGFP in supracellular actin cables increases significantly as the cables contract.

Цитати

"Live imaging of the TgT2[UbC:Lifeact-EGFP] quail has enabled the first high-resolution visualisation of the dynamics and behaviour of neural tube protrusions, and suggests a potential mechanical role for these structures in zippering of the neural folds."
"Our findings support the growing evidence that apical constriction driven by a ratchet-like mechanism is a common process shared by invertebrates and vertebrates."
"The increasing number and complexity of the multicellular rosettes during neural plate bending suggests that the persistence of these structures is higher than the transitory rosettes involved in tissue remodelling in Drosophila."

Ключові висновки, отримані з

The Lifeact-EGFP Quail: A New Avian Model For Studying Actin Dynamics In Vivo

by Alvarez,Y. D... о www.biorxiv.org 11-19-2023

https://www.biorxiv.org/content/10.1101/2023.11.19.567639v2

Глибші Запити

How could the Lifeact-EGFP quail model be used to investigate the interplay between actin dynamics and other cytoskeletal components, such as microtubules, during embryonic morphogenesis

The Lifeact-EGFP quail model provides a powerful tool for investigating the interplay between actin dynamics and other cytoskeletal components, such as microtubules, during embryonic morphogenesis. By utilizing live imaging techniques, researchers can simultaneously visualize the dynamics of actin filaments labeled with Lifeact-EGFP and microtubules labeled with appropriate markers. This allows for the observation of how these two cytoskeletal components interact and coordinate their activities during various morphogenetic processes.
One potential approach would be to perform dual-color live imaging using Lifeact-EGFP to label actin filaments and a fluorescent marker specific to microtubules. By tracking the movements and interactions of both cytoskeletal elements in real-time, researchers can gain insights into how actin and microtubules cooperate or compete to drive cellular behaviors such as cell migration, cell shape changes, and tissue morphogenesis. For example, observing the dynamics of actin-rich protrusions in coordination with microtubule-based structures like the mitotic spindle during cell division could provide valuable information on the mechanical forces and signaling pathways involved in these processes.
Furthermore, the Lifeact-EGFP quail model can be used in combination with pharmacological inhibitors or genetic manipulations targeting specific cytoskeletal components to dissect the functional roles of actin-microtubule crosstalk in embryonic development. By perturbing either actin or microtubule dynamics and observing the consequences on tissue morphogenesis, researchers can elucidate the cooperative or antagonistic relationships between these cytoskeletal elements.

What are the potential limitations or caveats of using a transgenic reporter line to study actin dynamics, and how could these be addressed

While the transgenic Lifeact-EGFP quail model offers significant advantages for studying actin dynamics in vivo, there are potential limitations and caveats that researchers should consider when using a transgenic reporter line for this purpose.

Overexpression artifacts: One concern with transgenic reporter lines is the possibility of overexpression artifacts, where the high levels of the fluorescent protein may alter the normal behavior of the labeled protein or its interacting partners. To address this, researchers can carefully optimize the expression levels of the transgene and validate the phenotypes observed with complementary approaches such as immunostaining or functional assays.

Cell type specificity: The expression of the Lifeact-EGFP transgene may not be restricted to specific cell types or tissues, leading to non-specific labeling and potential misinterpretation of results. To mitigate this, researchers can use cell type-specific promoters or enhancers to drive transgene expression in a more targeted manner, ensuring that the observed phenotypes are relevant to the specific cell populations of interest.

Temporal resolution: Live imaging of embryonic development requires high temporal resolution to capture dynamic processes such as cell migration, cell shape changes, and tissue morphogenesis. The speed of imaging and the duration of observation may be limited by the phototoxicity of the fluorescent protein and the developmental stage of the embryo. Researchers can optimize imaging parameters, such as laser power and acquisition settings, to minimize photodamage while maximizing the temporal resolution of the observations.

Quantitative analysis: Quantifying and analyzing the vast amount of live imaging data generated from the transgenic model can be challenging. Researchers need to employ robust image analysis tools and algorithms to extract meaningful quantitative information from the dynamic behaviors of actin structures in the developing embryo.

By addressing these limitations and caveats, researchers can maximize the utility of the transgenic Lifeact-EGFP quail model for studying actin dynamics during embryonic morphogenesis.

Given the insights into the role of supracellular actin cables and rosettes in neural tube formation, how might these structures contribute to the development or patterning of other organ systems

The insights gained from studying supracellular actin cables and rosettes in neural tube formation using the Lifeact-EGFP quail model have broader implications for understanding the development and patterning of other organ systems. These structures play crucial roles in tissue reorganization, mechanical force generation, and cell-cell interactions, which are fundamental processes in embryonic morphogenesis.

Organogenesis: Supracellular actin cables and rosettes are not unique to neural tube formation but are also involved in the development of other organ systems. For example, in the heart, these structures may contribute to the coordinated contraction of cardiac cells during heart tube formation and looping. By studying the dynamics of actin cables and rosettes in the context of heart development, researchers can uncover how these structures regulate tissue architecture and function in different organ systems.

Tissue mechanics: The formation and contraction of supracellular actin cables are essential for generating mechanical forces that drive tissue bending, folding, and elongation. In organs like the gut, lung, or kidney, these structures may play critical roles in shaping the organ architecture and establishing proper tissue organization. Understanding how actin cables and rosettes contribute to tissue mechanics in various organ systems can provide insights into the principles of tissue morphogenesis and homeostasis.

Disease implications: Dysregulation of actin dynamics and cytoskeletal organization is associated with various developmental disorders and diseases. Studying the role of supracellular actin cables and rosettes in organ development can shed light on the pathophysiology of conditions such as congenital malformations, cancer metastasis, and tissue regeneration. By elucidating the molecular mechanisms underlying these structures, researchers can identify potential therapeutic targets for treating cytoskeletal-related disorders.

Overall, the findings from investigating supracellular actin cables and rosettes in neural tube formation can be extrapolated to understand their contributions to the development and patterning of diverse organ systems, offering new insights into the mechanisms driving embryonic morphogenesis.