The shift to 3D growth during embryogenesis of kelp species, atlas of cell division and differentiation of Saccharina latissima.

In most organisms, 3D growth takes place at the onset of embryogenesis. In some brown algae, 3D growth occurs later in development, when the organism consists of several hundred cells. We studied the cellular events that take place when 3D growth is established in the embryo of the brown alga Saccharina, a kelp species. Semi-thin sections, taken from where growth shifts from 2D to 3D, show that 3D growth first initiates from symmetrical cell division in the monolayered lamina, and then is enhanced through a series of asymmetrical cell divisions in a peripheral monolayer of cells called the meristoderm. Then, daughter cells rapidly differentiate into cortical and medullary cells, characterised by their position, size and shape. In essence, 3D growth in kelps is based on a series of differentiation steps that occur rapidly after the initiation of a bilayered lamina, followed by further growth of the established differentiated tissues. Our study depicts the cellular landscape necessary to study cell-fate programming in the context of a novel mode of 3D growth in an organism phylogenetically distant from plants and animals.

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices.

The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen's health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device's operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen's cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

Targeted Laser Ablation in the Embryo of Saccharina latissima.

In Saccharina latissima, the embryo develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily distinguishable from its neighbors, and can be individually targeted. For decades, laser ablation has been used to study embryo development. Here, a protocol for cell-specific laser ablation was developed for early embryos of the brown alga S. latissima. The presented work includes: (1) the preparation of Saccharina embryos, with a description of the critical parameters, including culture conditions, (2) the laser ablation settings, and (3) the monitoring of the subsequent growth of the irradiated embryo using time-lapse microscopy. In addition, details are provided on the optimal conditions for transporting the embryos from the imaging platform back to the lab, which can profoundly affect subsequent embryo development. Algae belonging to the order Laminariales display embryogenesis patterns similar to Saccharina; this protocol can thus be easily transferred to other species in this taxon.

Design Principles of Branching Morphogenesis in Filamentous Organisms.

The radiation of life on Earth was accompanied by the diversification of multicellular body plans in the eukaryotic kingdoms Animalia, Plantae, Fungi and Chromista. Branching forms are ubiquitous in nature and evolved repeatedly in the above lineages. The developmental and genetic basis of branch formation is well studied in the three-dimensional shoot and root systems of land plants, and in animal organs such as the lung, kidney, mammary gland, vasculature, etc. Notably, recent thought-provoking studies combining experimental analysis and computational modeling of branching patterns in whole animal organs have identified global patterning rules and proposed unifying principles of branching morphogenesis. Filamentous branching forms represent one of the simplest expressions of the multicellular body plan and constitute a key step in the evolution of morphological complexity. Similarities between simple and complex branching forms distantly related in evolution are compelling, raising the question whether shared mechanisms underlie their development. Here, we focus on filamentous branching organisms that represent major study models from three distinct eukaryotic kingdoms, including the moss Physcomitrella patens (Plantae), the brown alga Ectocarpus sp. (Chromista), and the ascomycetes Neurospora crassa and Aspergillus nidulans (Fungi), and bring to light developmental regulatory mechanisms and design principles common to these lineages. Throughout the review we explore how the regulatory mechanisms of branching morphogenesis identified in other models, and in particular animal organs, may inform our thinking on filamentous systems and thereby advance our understanding of the diverse strategies deployed across the eukaryotic tree of life to evolve similar forms.

Alginates along the filament of the brown alga Ectocarpus help cells cope with stress.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus, we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation.

The brown algal mode of tip growth: Keeping stress under control.

Tip growth has been studied in pollen tubes, root hairs, and fungal and oomycete hyphae and is the most widely distributed unidirectional growth process on the planet. It ensures spatial colonization, nutrient predation, fertilization, and symbiosis with growth speeds of up to 800 µm h-1. Although turgor-driven growth is intuitively conceivable, a closer examination of the physical processes at work in tip growth raises a paradox: growth occurs where biophysical forces are low, because of the increase in curvature in the tip. All tip-growing cells studied so far rely on the modulation of cell wall extensibility via the polarized excretion of cell wall-loosening compounds at the tip. Here, we used a series of quantitative measurements at the cellular level and a biophysical simulation approach to show that the brown alga Ectocarpus has an original tip-growth mechanism. In this alga, the establishment of a steep gradient in cell wall thickness can compensate for the variation in tip curvature, thereby modulating wall stress within the tip cell. Bootstrap analyses support the robustness of the process, and experiments with fluorescence recovery after photobleaching (FRAP) confirmed the active vesicle trafficking in the shanks of the apical cell, as inferred from the model. In response to auxin, biophysical measurements change in agreement with the model. Although we cannot strictly exclude the involvement of a gradient in mechanical properties in Ectocarpus morphogenesis, the viscoplastic model of cell wall mechanics strongly suggests that brown algae have evolved an alternative strategy of tip growth. This strategy is largely based on the control of cell wall thickness rather than fluctuations in cell wall mechanical properties.

Gazing at Cell Wall Expansion under a Golden Light.

In plants, cell growth is constrained by a stiff cell wall, at least this is the way textbooks usually present it. Accordingly, many studies have focused on the elasticity and plasticity of the cell wall as prerequisites for expansion during growth. With their specific evolutionary history, cell wall composition, and environment, brown algae present a unique configuration offering a new perspective on the involvement of the cell wall, viewed as an inert material yet with intrinsic mechanical properties, in growth. In light of recent findings, we explore here how much of the functional relationship between cell wall chemistry and intrinsic mechanics on the one hand, and growth on the other hand, has been uncovered in brown algae.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Furthering knowledge of seaweed growth and development to facilitate sustainable aquaculture.

Macroalgae (seaweeds) are the subject of increasing interest for their potential as a source of valuable, sustainable biomass in the food, feed, chemical and pharmaceutical industries. Compared with microalgae, the pace of knowledge acquisition in seaweeds is slower despite the availability of whole-genome sequences and model organisms for the major seaweed groups. This is partly a consequence of specific hurdles related to the large size of these organisms and their slow growth. As a result, this basic scientific field is falling behind, despite the societal and economic importance of these organisms. Here, we argue that sustainable management of seaweed aquaculture requires fundamental understanding of the underlying biological mechanisms controlling macroalgal life cycles - from the production of germ cells to the growth and fertility of the adult organisms - using diverse approaches requiring a broad range of technological tools. This Viewpoint highlights several examples of basic research on macroalgal developmental biology that could enable the step-changes which are required to adequately meet the demands of the aquaculture sector.

Morphoelasticity in the development of brown alga Ectocarpus siliculosus: from cell rounding to branching.

A biomechanical model is proposed for the growth of the brown alga Ectocarpus siliculosus Featuring ramified uniseriate filaments, this alga has two modes of growth: apical growth and intercalary growth with branching. Apical growth occurs upon the mitosis of a young cell at one extremity and leads to a new tip cell followed by a cylindrical cell, whereas branching mainly occurs when a cylindrical cell becomes rounded and swells, forming a spherical cell. Given the continuous interplay between cell growth and swelling, a poroelastic model combining osmotic pressure and volumetric growth is considered for the whole cell, cytoplasm and cell wall. The model recovers the morphogenetic transformations of mature cells: transformation of a cylindrical shape into spherical shape with a volumetric increase, and then lateral branching. Our simulations show that the poro-elastic model, including the Mooney-Rivlin approach for hyper-elastic materials, can correctly reproduce the observations. In particular, branching appears to be a plasticity effect due to the high level of tension created after the increase in volume of mature cells.

Surfing amongst Oil-Tankers: Connecting Emerging Research Fields to the Current International Landscape.

The COST Action Phycomorph (FA1406) was initiated in 2015 from a handful of academic researchers, and now joins together 19 European countries and nine international partners. Phycomorph's goal is to coordinate and develop research on developmental biology in macroalgae. This is an ambitious project, as the related scientific community is small, the concepts are complex, and there is currently limited knowledge of these organisms and there are few technologies to study them. Here we report the first step in achieving this enterprise, the creation of the Phycomorph network. We share the associated strengths, pitfalls, and prospects for setting up the network in the hope that this might guide similar efforts in other fields.

Production of genetically and developmentally modified seaweeds: exploiting the potential of artificial selection techniques.

Plant feedstock with specific, modified developmental features has been a quest for centuries. Since the development and spread of agriculture, there has been a desire for plants producing disproportionate-or more abundant and more nutritional-biomass that meet human needs better than their native counterparts. Seaweed aquaculture, targeted for human consumption and the production of various raw materials, is a rapidly expanding field and its stakeholders have increasing vested interest for cost-effective and lucrative seaweed cultivation processes. Thus, scientific research on seaweed development is particularly timely: the potential for expansion of seaweed cultivation depends on the sector's capacity to produce seaweeds with modified morphological features (e.g., thicker blades), higher growth rates or delayed (or even no) fertility. Here, we review the various technical approaches used to modify development in macroalgae, which have attracted little attention from developmental biologists to date. Because seaweed (or marine macroalgae) anatomy is much less complex than that of land plants and because seaweeds belong to three different eukaryotic phyla, the mechanisms controlling their morphogenesis are key to understanding their development. Here, we present efficient sources of developmentally and genetically modified seaweeds-somatic variants, artificial hybrids and mutants-as well as the future potential of these techniques.

Localization of causal locus in the genome of the brown macroalga Ectocarpus: NGS-based mapping and positional cloning approaches.

Mutagenesis is the only process by which unpredicted biological gene function can be identified. Despite that several macroalgal developmental mutants have been generated, their causal mutation was never identified, because experimental conditions were not gathered at that time. Today, progresses in macroalgal genomics and judicious choices of suitable genetic models make mutated gene identification possible. This article presents a comparative study of two methods aiming at identifying a genetic locus in the brown alga Ectocarpus siliculosus: positional cloning and Next-Generation Sequencing (NGS)-based mapping. Once necessary preliminary experimental tools were gathered, we tested both analyses on an Ectocarpus morphogenetic mutant. We show how a narrower localization results from the combination of the two methods. Advantages and drawbacks of these two approaches as well as potential transfer to other macroalgae are discussed.

The green seaweed Ulva: a model system to study morphogenesis.

Green macroalgae, mostly represented by the Ulvophyceae, the main multicellular branch of the Chlorophyceae, constitute important primary producers of marine and brackish coastal ecosystems. Ulva or sea lettuce species are some of the most abundant representatives, being ubiquitous in coastal benthic communities around the world. Nonetheless the genus also remains largely understudied. This review highlights Ulva as an exciting novel model organism for studies of algal growth, development and morphogenesis as well as mutualistic interactions. The key reasons that Ulva is potentially such a good model system are: (i) patterns of Ulva development can drive ecologically important events, such as the increasing number of green tides observed worldwide as a result of eutrophication of coastal waters, (ii) Ulva growth is symbiotic, with proper development requiring close association with bacterial epiphytes, (iii) Ulva is extremely developmentally plastic, which can shed light on the transition from simple to complex multicellularity and (iv) Ulva will provide additional information about the evolution of the green lineage.

Laser capture microdissection in Ectocarpus siliculosus: the pathway to cell-specific transcriptomics in brown algae.

Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.

Brown algal morphogenesis: atomic force microscopy as a tool to study the role of mechanical forces.

Over the last few years, a growing interest has been directed toward the use of macroalgae as a source of energy, food and molecules for the cosmetic and pharmaceutical industries. Besides this, macroalgal development remains poorly understood compared to other multicellular organisms. Brown algae (Phaeophyceae) form a monophyletic lineage of usually large multicellular algae which evolved independently from land plants. In their environment, they are subjected to strong mechanical forces (current, waves, and tide), in response to which they modify rapidly and reversibly their morphology. Because of their specific cellular features (cell wall composition, cytoskeleton organization), deciphering how they cope with these forces might help discover new control mechanisms of cell wall softening and cellulose synthesis. Despite the current scarcity in knowledge on brown algal cell wall dynamics and protein composition, we will illustrate, in the light of methods adapted to Ectocarpus siliculosus, to what extent atomic force microscopy can contribute to advance this field of investigation.

Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus.

We used an in silico approach to predict microRNAs (miRNAs) genome-wide in the brown alga Ectocarpus siliculosus. As brown algae are phylogenetically distant from both animals and land plants, our approach relied on features shared by all known organisms, excluding sequence conservation, genome localization and pattern of base-pairing with the target. We predicted between 500 and 1500 miRNAs candidates, depending on the values of the energetic parameters used to filter the potential precursors. Using quantitative polymerase chain reaction assays, we confirmed the existence of 22 miRNAs among 72 candidates tested, and of 8 predicted precursors. In addition, we compared the expression of miRNAs and their precursors in two life cycle states (sporophyte, gametophyte) and under salt stress. Several miRNA precursors, Argonaute and DICER messenger RNAs were differentially expressed in these conditions. Finally, we analyzed the gene organization and the target functions of the predicted candidates. This showed that E. siliculosus miRNA genes are, like plant miRNA genes, rarely clustered and, like animal miRNA genes, often located in introns. Among the predicted targets, several widely conserved functional domains are significantly overrepresented, like kinesin, nucleotide-binding/APAF-1, R proteins and CED-4 (NB-ARC) and tetratricopeptide repeats. The combination of computational and experimental approaches thus emphasizes the originality of molecular and cellular processes in brown algae.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

Culture methods and mutant generation in the filamentous brown algae Ectocarpus siliculosus.

Ectocarpus siliculosus is a small filamentous alga that has recently emerged as the new model for fundamental research on brown algae. Here, we describe the basic culture protocols for propagating and collecting E. siliculosus material that can then be used in all types of molecular biology, biochemistry and cell biology techniques. In addition, procedures for carrying out genetic experiments (generation of mutants and genetic segregation analyses) on E. siliculosus are described.