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At least 343 records · Page 19Linked to original sources

bioRxiv2023

Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.

synthetic biology↗

bioRxiv2023

New resources for environmental genomics in the wild Mediterranean grass B. distachyon

AO_SCPLOWBSTRACTC_SCPLOWClosely related to economically important crops, the grass Brachypodium distachyon has been originally established as a pivotal species for grass genomics but more recently flourished as a model for developmental biology. Grasses encompass more than 10,000 species and cover more than 40% of the world land area from tropical to temperate regions. Given that grasses also supply about a fifth of the worlds dietary protein as cereal grains, unlocking the sources of phenotypic variation in B. distachyon is hence of prime interest in fundamental and applied research in agronomy, ecology and evolution. We present here the B. distachyon diversity panel, which encompasses 332 fully sequenced accessions covering the whole species distribution from Spain to Iraq. By combining population genetics, niche modeling and landscape genomics, we suggest that B. distachyon recolonized Europe and the Middle East following the last glacial maximum. Consequently, the species faced new environmental conditions which led to clear associations between bioclimatic variables and genetic factors as well as footprints of positive selection in the genome. Altogether, this genomic resource offers a powerful alternative to Arabidopsis thaliana to investigate the genetic bases of adaptation and phenotypic plasticity in plants and more specifically in monocots.

ecology↗

bioRxiv2023

An inducible germ cell ablation chicken model for high-grade germline chimeras

Chicken embryos are a powerful and widely used animal model in developmental biology studies. After the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) after genetic modifications. However, the low inheritance caused by the competition between host germ cells and the transferred ones is the most common complication and largely reduces the production efficiency in this way. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which NTR produces cytotoxic alkylating agents from administered Mtz, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus is used to drive the expression of the NTR gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system germ cell-Specific AutonoMoUs RemovAl Induction (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

bioengineering↗

bioRxiv2023

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 {micro}m laterally, 3.3 {micro}m axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

neuroscience↗

bioRxiv2023

Assembly archetypes in ecological communities

An instrumental discovery in comparative and developmental biology is the existence of assembly archetypes that synthesize the vast diversity of organisms body plans--from legs and wings to human arms--into simple, interpretable, and general design principles. Here we combine a novel mathematical formalism based on Category Theory with experimental data to show that similar "assembly archetypes" exist at the larger organization scale of ecological communities when assembling a species pool across diverse environmental contexts, particularly when species interactions are highly structured. We applied our formalism to clinical data discovering two assembly archetypes that differentiate between healthy and unhealthy human gut microbiota. The concept of assembly archetypes and the methods to synthesize them can pave the way to discovering the general assembly principles of the ecological communities we observe in nature.

ecology↗

bioRxiv2023

Liver-specification of human iPSC-derived endothelial cells transplanted into mouse liver

Liver sinusoidal endothelial cells (LSECs) play an important role in liver development, regeneration and pathophysiology, but the differentiation process that generates their unique tissue-specific phenotype is poorly understood and difficult to study as primary cells are only available in limited quantities. To address this, we hypothesised that human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) can produce hiPSC-derived LSECs upon transplantation into the livers of Fah-/-/Rag2-/-/Il2rg-/-mice, and serve as a model to study LSEC specification. Progressive and long-term repopulation of the liver vasculature was observed, as iECs expanded along the sinusoids that run between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptome of transplanted cells at time-points between 1 and 12 weeks were compared against primary human adult LSECs, which demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS play a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process include EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contain zonal subpopulations with a region-specific phenotype. Collectively, this study confirms that hiPSC can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs.

cell biology↗

bioRxiv2023

Automatic design of gene regulatory mechanisms for spatial pattern formation

Synthetic developmental biology aims to engineer gene regulatory mechanisms (GRMs) for understanding and producing desired multicellular patterns and shapes. However, designing GRMs for spatial patterns is a current challenge due to the nonlinear interactions and feedback loops in genetic circuits. Here we present a methodology to automatically design GRMs that can produce any given spatial pattern. The proposed approach uses two orthogonal morphogen gradients acting as positional information signals in a multicellular tissue area or culture, which constitutes a continuous field of engineered cells implementing the same designed GRM. To efficiently design both the circuit network and the interaction mechanisms--including the number of genes necessary for the formation of the target pattern--we developed an automated algorithm based on high-performance evolutionary computation. The tolerance of the algorithm can be configured to design GRMs that are either simple to produce approximate patterns or complex to produce precise patterns. We demonstrate the approach by automatically designing GRMs that can produce a diverse set of synthetic spatial expression patterns by interpreting just two orthogonal morphogen gradients. The proposed framework offers a versatile approach to systematically design and discover pattern-producing genetic circuits.

systems biology↗

bioRxiv2023

Control of cell fate specification and patterning by an ancestral microRNA

The formation of an organized body requires the establishment and maintenance of cells with structural and functional distinctive characteristics. A central question in developmental biology is how changes in the regulation of genes drive cell specification and patterning1. microRNAs (miRNAs) are small non-coding RNAs that regulate development through mRNA cleavage and/or translational repression2. In plants, miRNAs regulate key aspects including growth, development, stem cell maintenance, vegetative phase change, leaf morphogenesis, floral organ formation and flowering time3. Biogenesis of plant miRNAs depends on the activity of DICER-LIKE 1 (DCL1), an RNase type III endonuclease that processes double stranded RNA to give rise to mature miRNAs 4. The genomes of todays flora contain at least one bona fide copy of DCL1 5,6. Using Marchantia polymorpha -a model bryophyte that allows comparative approaches to infer characteristics of the ancestral land plant-, we demonstrate that MpDCL1a is required for the biogenesis of miRNAs and uncovered a central role for miR166/Homeodomain Zipper Class III-regulated auxin synthesis in the specification of cell identity, patterning, meristem function, laminar expansion and the development of the body in the last common ancestor of extant land plants.

plant biology↗

bioRxiv2023

scEGOT: Single-cell trajectory inference framework based on entropic Gaussian mixture optimal transport

Time-series scRNA-seq data have opened a door to elucidate cell differentiation, and in this context, the optimal transport theory has been attracting much attention. However, there remain critical issues in interpretability and computational cost. We present scEGOT, a comprehensive framework for single-cell trajectory inference, as a generative model with high interpretability and low computational cost. Applied to the human primordial germ cell-like cell (PGCLC) induction system, scEGOT identified the PGCLC progenitor population and bifurcation time of segregation. Our analysis shows TFAP2A is insufficient for identifying PGCLC progenitors, requiring NKX1-2. Additionally, MESP1 and GATA6 are also crucial for PGCLC/somatic cell segregation. These findings shed light on the mechanism that segregates PGCLC from somatic lineages. Notably, not limited to scRNA-seq, scEGOTs versatility can extend to general single-cell data like scATAC-seq, and hence has the potential to revolutionize our understanding of such datasets and, thereby also, developmental biology.

bioinformatics↗

bioRxiv2023

Automated Stimulation and Long-Term Remote Monitoring of Multi-Plex Microfluidic Organoid Chips

Developing a novel microfluidic organoid system required many experiments and iterations due to lack of knowledge about the relevant developmental biology. Collecting data on the developing organoids quickly escalated into a bottleneck as high throughput and long term culture resulted in a rapidly increasing number of specimens being observed. Commercially available automated microscope systems exist, but were either too expensive or not appropriate, and could not be modified. To satisfy the increasing need for automated data collection, a custom robotic system was developed to collect data from within a standard incubator. An X-Y belt driven gantry was designed with an architecture chosen to balance high accuracy, low cost, speed, and range of motion. Focus control was implemented with dual miniature leadscrews. A linear sliding mechanism was used to switch between two microscope objectives. 3D printed chip attachments were designed to implement illuminators for bright field imaging, and electrodes for stimulating the cardiac organoids. A fluorescent filter block was designed using a 3D printed piece to hold optical components, and a multi-band filter set that allowed for three color fluorescence without moving parts. A pulley driven tilting stage gravitationally biased the organoids during development. In order to ensure accurate image collection despite the inevitable position shifting of the chips, an image processing pipeline was developed for locating organoids using geometrical microfluidic chip features. The resulting robotic system automated imaging data collection on organoids and electrical and mechanical stimulation, in addition to being modifiable for future projects.

bioengineering↗

bioRxiv2023

Open-source, high-throughput targeted in-situ transcriptomics for developmental biologists

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in-situ expression maps of hundreds of genes. We exploit direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing. We validate the method across a number of species, and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to highthroughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

molecular biology↗

bioRxiv2023

Fine-grained modelling of ATP dependence of decision-making capacity in genetic regulatory networks

Cellular decision-making is fundamental to life, from developmental biology to environmental responses and antimicrobial resistance. Many regulatory processes that drive cellular decisions rely on gene expression, which requires energy in the form of ATP. As even genetically identical cells can have dramatically different ATP levels, bioenergetic status can be an important source of variability in cellular decision-making. Existing studies have investigated this energy dependence but often use coarse-grained modelling approaches (which are not always readily connected to the underlying molecular processes of gene regulation). Here we use a fine-grained mathematical model of gene expression in a two-gene decision-making regulatory network to explore cellular decision-making capacity as energy availability varies. We simulate both a deterministic model, to explore the emergence of different cell fate attractors as ATP levels vary, and a stochastic case to explore how ATP influences the noisy dynamics of stochastic cell decision-making. Higher energy levels typically support increased decision-making capacity (higher numbers of, and more separated, cell states that can be selected), and the fine-grained modelling reveals some differences in behaviour from previous coarse-grained modelling approaches.

systems biology↗

bioRxiv2023

Allocator is a graph neural network-based framework for mRNA subcellular localization prediction

MotivationThe asymmetrical distribution of expressed mRNAs tightly controls the precise synthesis of proteins within human cells. This non-uniform distribution, a cornerstone of developmental biology, plays a pivotal role in numerous cellular processes. To advance our comprehension of gene regulatory networks, it is essential to develop computational tools for accurately identifying the subcellular localizations of mRNAs. However, considering multi-localization phenomena remains limited in existing approaches, with none considering the influence of RNAs secondary structure. ResultsIn this study, we propose Allocator, a multi-view parallel deep learning framework that seamlessly integrates the RNA sequence-level and structure-level information, enhancing the prediction of mRNA multi-localization. The Allocator models equip four efficient feature extractors, each designed to handle different inputs. Two are tailored for sequence-based inputs, incorporating multilayer perceptron and multi-head self-attention mechanisms. The other two are specialized in processing structure-based inputs, employing graph neural networks. Benchmarking results underscore Allocators superiority over state-of-the-art methods, showcasing its strength in revealing intricate localization associations. AvailabilityThe webserver of Allocator is available at http://Allocator.unimelb-biotools.cloud.edu.au; the source code and datasets are available at https://github.com/lifuyi774/Allocator

bioinformatics↗

bioRxiv2023

Single-nucleus RNA-sequencing reveals novel potential mechanisms of ovarian insufficiency in 45,X Turner Syndrome

Study questionCan single-nuclei and bulk RNA sequencing technologies be used to elucidate novel mechanisms of ovarian insufficiency in Turner Syndrome (TS)? Summary answerUsing single-nucleus and bulk RNA sequencing approaches, we identified novel potential pathogenic mechanisms underlying ovarian insufficiency in TS including and beyond X chromosome haploinsufficiency. What is known alreadyTurner syndrome (TS) is the most common genetic cause of Primary Ovarian Insufficiency (POI) in humans. Morphological analyses of human fetal 45,X ovaries have demonstrated fewer germ cells and marked apoptosis established by 15-20 weeks post conception (wpc); however, we do not understand why POI develops mechanistically in the first instance. Study design, size, durationSingle-nucleus RNA sequencing (snRNA-seq): two 46,XX and two 45,X (TS) human fetal ovaries at 12-13 wpc. Bulk RNA sequencing: 19 human fetal ovary, 20 fetal testis, and 8 fetal control tissue (n=47 total samples; Carnegie Stage 22-16wpc). Participants/materials, setting, methodsTo identify novel potential mechanisms of ovarian insufficiency in TS and to characterise X chromosome gene expression in the 45,X ovary, we performed snRNA-seq of peri-meiotic 46,XX (n=2) and 45,X (n=2) fetal ovaries at 12-13 weeks post conception (wpc); and 2) a bulk RNA sequencing time-series analysis of fetal ovary, testis, and control samples across four developmental timepoints. Main results and the role of chanceGerm and somatic cell subpopulations were mostly shared across 46,XX and 45,X ovaries, aside from a 46XX-specific/45,X-depleted cluster of oogonia ("synaptic oogonia") containing genes with functions relating to sex chromosome synapsis; histone modification; intracellular protein regulation and chaperone systems. snRNA-seq enabled accurate cell counting localised to individual cell clusters; the 45,X ovary has fewer germ cells than the 46,XX ovary in every germ cell subpopulation, confirmed by histopathological analysis. The normal sequence of X-chromosome inactivation and reactivation is disrupted in 45,X ovaries; XIST was not expressed in 45,X somatic cells but was present in germ cell clusters, albeit with lower expression than in corresponding 46,XX clusters. The 45,X ovary has a globally abnormal transcriptome, with low expression of genes with proteostasis functions (RSP4X); cell cycle progression (BUB1B); and OXPHOS mitochondrial energy production (COX6C, ATP11C). Genes with higher expression in 45,X cell populations were enriched for apoptotic functions (e.g., NR4A1). Limitations, reasons for cautionLimitations include the relatively small sample size of the snRNA-seq analysis and the focus on a fixed meiotic timepoint which may overlook a dynamic process over time. Wider implications of the findingsWe characterise the human fetal peri-meiotic 45,X ovary at single-cell resolution and offer insights into novel pathogenic mechanisms underlying ovarian insufficiency in TS. Although asynapsis due to X chromosome haploinsufficiency likely plays a significant role, these data suggest meiotic failure and ovarian insufficiency may be a combinatorial process characterised by periods of vulnerability throughout early 45,X germ cell development Study funding/competing interest(s)This research was funded in whole, or in part, by the Wellcome Trust Grants 216362/Z/19/Z to SMcG-B and 209328/Z/17/Z to JCA. Human fetal material was provided by the Joint MRC/Wellcome Trust (Grant MR/R006237/1) Human Developmental Biology Resource (http://www.hdbr.org). Research at UCL Great Ormond Street Institute of Child Health is supported by the National Institute for Health Research, Great Ormond Street Hospital Biomedical Research Centre (grant IS-BRC-1215-20012).

genomics↗

bioRxiv2024

An iPSC-derived small intestine-on-chip with self-organizing epithelial, mesenchymal and neural cells

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their closed topology and relative immature state limits their applications. Here we use organ-on-chip technology to develop a hiPSC-derived intestinal barrier with apical and basolateral access in a more physiological in vitro microenvironment. To replicate growth factor gradients along the crypt- villus axis, we locally exposed the cells to expansion and differentiation media. In these conditions, intestinal epithelial cells self-organize into villus-like folds with physiological barrier integrity and myofibroblast and neural subtypes emerge and form a layer in the bottom channel underneath the epithelial tissue. The growth factor gradients efficiently balance dividing and mature cell types and induce an intestinal epithelial composition, including absorptive and secretory lineages, resembling the composition of the human adult small intestine. The result is a well-characterized hiPSC-derived intestine-on-chip system that can facilitate personalized studies on physiological processes and therapy development in the human small intestine.

cell biology↗

bioRxiv2024

Three-dimensional Label-Free Measurements of Adipocyte Differentiation and Lipid Droplet Dynamics

The visualization and tracking of adipocytes and their lipid droplets (LDs) during differentiation are pivotal in developmental biology and regenerative medicine studies. Traditional staining or labeling methods, however, pose significant challenges due to their labor-intensive sample preparation, potential disruption of intrinsic cellular physiology, and limited observation timeframe. This study introduces a novel method for long-term visualization and quantification of biophysical parameters of LDs in unlabeled adipocytes, utilizing the refractive index (RI) distributions of LDs and cells. We employ low-coherence holotomography (HT) to systematically investigate and quantitatively analyze the 42-day redifferentiation process of fat cells into adipocytes. This technique yields three-dimensional, high-resolution refractive tomograms of adipocytes, enabling precise segmentation of LDs based on their elevated RI values. Subsequent automated analysis quantifies the mean concentration, volume, projected area, and dry mass of individual LDs, revealing a gradual increase corresponding with adipocyte maturation. Our findings demonstrate that HT is a potent tool for non-invasively monitoring live adipocyte differentiation and analyzing LD accumulation. This study, therefore, offers valuable insights into adipogenesis and lipid research, establishing HT and image-based analysis as a promising approach in these fields.

biochemistry↗

bioRxiv2024

Multiple Displacement Amplification Facilitates SMRT Sequencing of Microscopic Animals and the Genome of the Gastrotrich Lepidodermella squamata (Dujardin, 1841)

BackgroundObtaining adequate DNA for long-read genome sequencing remains a roadblock to producing contiguous genomes from small-bodied organisms. Multiple displacement amplification (MDA) leverages Phi29 DNA polymerase to produce micrograms of DNA from picograms of input. Few genomes have been generated using this approach, due to concerns over biases in amplification related to GC and repeat content and chimera production. Here, we explored the utility of MDA for generating template DNA for PacBio HiFi sequencing using Caenorhabditis elegans (Nematoda) and Lepidodermella squamata (Gastrotricha). ResultsHiFi sequencing of libraries prepared from MDA DNA produced highly contiguous and complete genomes for both C. elegans (102 Mbp assembly; 336 contigs; N50 = 868 Kbp; L50 = 39; BUSCO_nematoda: S:92.2%, D:2.7%) and L. squamata (122 Mbp assembly; 157 contigs; N50 = 3.9 Mb; L50 = 13; BUSCO_metazoa: S: 78.0%, D: 2.8%). Amplified C. elegans reads mapped to the reference genome with a rate of 99.92% and coverage of 99.75% with just one read (of 708,811) inferred to be chimeric. Coverage uniformity was nearly identical for reads from MDA DNA and reads from pooled worm DNA when mapped to the reference genome. The genome of Lepidodermella squamata, the first of its phylum, was leveraged to infer the phylogenetic position of Gastrotricha, which has long been debated, as the sister taxon of Platyhelminthes. ConclusionsThis methodology will help generate contiguous genomes of microscopic taxa whose body size precludes standard long-read sequencing. L. squamata is an emerging model in evolutionary developmental biology and this genome will facilitate further work on this species.

genomics↗

bioRxiv2024

Understanding Regeneration: investigating the apical notch and meristem re-emergence in Marchantia polymorpha

Meristems are the growth centres of plants, and fundamental in understanding plant development, morphogenesis and vegetative propagation. Across all plant groups, the phytohormone auxin controls meristem maintenance, represses the emergence of new meristems (apical dominance) and mediates cellular reprogramming when new meristems regenerate following removal of existing meristems. The liverwort Marchantia produces clonal propagules (gemmae) featuring two apical notches that develop into functional meristems. This presents a tractable experimental system to study meristem developmental biology. I used laser ablation microscopy to precisely disrupt cells in and around the developing pre-meristem in the apical notches of germinating gemma, finding that the first cell row is indispensable. Within this layer, a contiguous quorum of stem cells is required for activity. Apical notches reorientate in response to damage, demonstrating that the apical notch stem cells act as a communicating population. Feedback from the stem cell population is necessary to maintain notch activity and generate the notch apex. These experiments show communication between notches and regenerating meristems. The apical dominance signal represses cell division and requires both sources and sinks, features of auxin-mediated communication. Central regions of the gemma could transmit these apical dominance signals, but the tissues of the gemma periphery could not. I present a model of Marchantia gemma and apical notch organization, involving intra-, inter-and extra-notch communication. This provides a framework for further study of meristem formation, communication and maintenance in Marchantia and improving knowledge of plant meristems more generally. Significance StatementMeristems are the growth centres of plants. Understanding how meristems function, how existing meristems stop new meristems emerging (apical dominance) and how cellular reprogramming regenerates meristems is central to understanding plant development. The model plant Marchantia offers a streamlined system whose evolutionary position makes its biology relevant to all plants. Marchantia apical notches display proliferative, regenerative and dominance behaviour typical of meristems. I used laser ablation to precisely disrupt cells in and around the developing pre-meristematic apical notch of the germinating Marchantia gemma, producing an improved model of Marchantia apical notch organization involving intra-and inter-notch communication characteristic of the phytohormone auxin. This will inform future Marchantia research and provides a simplified experimental framework to study more complex meristematic processes.

plant biology↗