a-robust-reproducible-protocol-for-neural-tube-organoid-generation

A Robust and Reproducible Protocol for Neural Tube Organoid Generation from Single Mouse Embryonic Stem Cells

The development of mammals is a highly complex process, characterized by the necessity for precise concentration- and time-dependent signaling for correct ...

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA)

Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here, we describe 2Eci (2nd generation ethyl cinnamate-based clearing), which can be used to clear a wide range of tissues in several species, including human organoids, , zebrafish, axolotl and , in as little as 1-5 days, while preserving a broad range of fluorescent proteins, including GFP, mCherry, Brainbow and Alexa-conjugated fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability.

Broad applicability of a streamlined ethyl cinnamate-based clearing procedure.

Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.

A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2 clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.

Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition.

Regeneration is a heroic biological process that restores tissue architecture and function in the face of day-to-day cell loss or the aftershock of injury. Capacities and mechanisms for regeneration can vary widely among species, organs, and injury contexts. Here, we describe "hallmarks" of regeneration found in diverse settings of the animal kingdom, including activation of a cell source, initiation of regenerative programs in the source, interplay with supporting cell types, and control of tissue size and function. We discuss these hallmarks with an eye toward major challenges and applications of regenerative biology.

Hallmarks of regeneration.

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue-patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signaling center that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal-ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridization and mathematical modeling, we first quantified the behaviors of dorsal-ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labeled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signaling center in the axolotl spinal cord.

Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord.

Fibrosis remains a major unmet medical need. Simplifying principles are needed to better understand fibrosis and to yield new therapeutic approaches. Fibrosis is driven by myofibroblasts that interact with macrophages. A mathematical cell-circuit model predicts two types of fibrosis: hot fibrosis driven by macrophages and myofibroblasts and cold fibrosis driven by myofibroblasts alone. Testing these concepts in cardiac fibrosis resulting from myocardial infarction (MI) and heart failure (HF), we revealed that acute MI leads to cold fibrosis whereas chronic injury (HF) leads to hot fibrosis. MI-driven cold fibrosis is conserved in pigs and humans. We computationally identified a vulnerability of cold fibrosis: the myofibroblast autocrine growth factor loop. Inhibiting this loop by targeting TIMP1 with neutralizing antibodies reduced myofibroblast proliferation and fibrosis post-MI in mice. Our study demonstrates the utility of the concepts of hot and cold fibrosis and the feasibility of a circuit-to-target approach to pinpoint a treatment strategy that reduces fibrosis. A record of this paper's transparent peer review process is included in the supplemental information.

Cold and hot fibrosis define clinically distinct cardiac pathologies.

Axolotls are amphibian models for studying nervous system evolution, development, and regeneration. Tools to visualize and manipulate cells of the axolotl nervous system with high-efficiency, spatial and temporal precision are therefore greatly required. Recombinant adeno-associated viruses (AAVs) are frequently used for in vivo gene transfer of the nervous system but virus-mediated gene delivery to the axolotl nervous system has not yet been described. Here, we demonstrate the use of AAVs for efficient gene transfer within the axolotl brain, the spinal cord, and the retina. We show that serotypes AAV8, AAV9, and AAVPHP.eB are suitable viral vectors to infect both excitatory and inhibitory neuronal populations of the axolotl brain. We further use AAV9 to trace retrograde and anterograde projections between the retina and the brain and identify a cell population projecting from the brain to the retina. Together, our work establishes AAVs as a powerful tool to interrogate neuronal organization in the axolotl.

Adeno-associated viruses for efficient gene expression in the axolotl nervous system.

The axolotl salamander model has broad utility for regeneration studies, but this model is limited by a lack of efficient cell-culture-based tools. The Axolotl Limb-1 (AL-1) fibroblast line, the only available immortalized axolotl cell line, was first published over 20 years ago, but many established molecular biology techniques, such as lipofectamine transfection, CRISPR-Cas9 mutagenesis, and antibiotic selection, work poorly or remain untested in AL-1 cells. Innovating technologies to manipulate AL-1 cells in culture and study their behavior following transplantation into the axolotl will complement in-vivo studies, decrease the number of animals used, and enable the faster, more streamlined investigation of regenerative biology questions. Here, we establish transfection, mutagenesis, antibiotic selection, and in-vivo transplantation techniques in axolotl AL-1 cells. These techniques will enable efficient culture with AL-1 cells and guide future tool development for the culture and manipulation of other salamander cell lines.

Optimized toolkit for the manipulation of immortalized axolotl fibroblasts.

The amputation of a salamander limb triggers anterior and posterior connective tissue cells to form distinct signalling centres that together fuel regeneration. Anterior and posterior identities are established during development and are thought to persist for the whole life in the form of positional memory. However, the molecular basis of positional memory and whether positional memory can be altered remain unknown. Here, we identify a positive-feedback loop that is responsible for posterior identity in the limb of an axolotl (Ambystoma mexicanum). Posterior cells express residual Hand2 transcription factor from development, and this primes them to form a Shh signalling centre after limb amputation. During regeneration, Shh signalling is also upstream of Hand2 expression. After regeneration, Shh is shut down but Hand2 is sustained, safeguarding posterior memory. We used this regeneration circuitry to convert anterior cells to a posterior-cell memory state. Transient exposure of anterior cells to Shh during regeneration kick-started an ectopic Hand2-Shh loop, leading to stable Hand2 expression and lasting competence to express Shh. Our results implicate positive-feedback in the stability of positional memory and reveal that positional memory is reprogrammed more easily in one direction (anterior to posterior) than in the other. Modifying positional memory in regenerative cells changes their signalling outputs, which has implications for tissue engineering.

Molecular basis of positional memory in limb regeneration.

The piRNA pathway protects animal germlines from active transposons. Mammals employ a cytoplasmic pathway to destroy transposon transcripts during germline reprogramming. This post-transcriptional mechanism is ancient and found throughout the animal kingdom. A nuclear piRNA pathway mediates transposon DNA re-methylation, which is believed to be bespoke to mammals. However, when exactly piRNA-directed DNA methylation evolved remains unknown. We found that a mammalian-like piRNA pathway evolved early in tetrapod evolution and is found and expressed in its current configuration in the axolotl salamander. Analysis of axolotl testes and oocytes revealed diverse repertoires of piRNAs and pervasive post-transcriptional targeting of young transposons. We identified high levels of genome methylation in axolotl spermatozoa, with full-length transposons being heavily methylated. Our findings reveal that the mammalian nuclear piRNA pathway has ancient vertebrate origins, and it has likely been safeguarding the germline throughout most of tetrapod evolution. Thus, the emergence of piRNA-directed DNA methylation is a pivotal epigenetic evolutionary event that may have laid the foundation for germline reprogramming and genomic imprinting.

Article	Year
Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development (Cambridge, England)\| PubMed ID: 30665888	2019
A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity. Science advances\| PubMed ID: 32523996	2020
Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition. Developmental cell\| PubMed ID: 38776925	2024
Hallmarks of regeneration. Cell stem cell\| PubMed ID: 39163854	2024
Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord. Development, growth & differentiation\| PubMed ID: 39387203	2024
Cold and hot fibrosis define clinically distinct cardiac pathologies. Cell systems\| PubMed ID: 39970910	2025
Adeno-associated viruses for efficient gene expression in the axolotl nervous system. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 40042904	2025
Optimized toolkit for the manipulation of immortalized axolotl fibroblasts. Methods (San Diego, Calif.)\| PubMed ID: 40187387	2025
Molecular basis of positional memory in limb regeneration. Nature\| PubMed ID: 40399677	2025
A mammalian-like piRNA pathway in Axolotl reveals the origins of piRNA-directed DNA methylation. The EMBO journal\| PubMed ID: 41233617	2025

Elly M. Tanaka

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