Bernard Billoud

Auxin Metabolism and Function in the Multicellular Brown Alga Ectocarpus siliculosus

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.

ETOILE Regulates Developmental Patterning in the Filamentous Brown Alga Ectocarpus siliculosus

By means of a combination of experimental and modeling approaches applied to the hyperbranching mutant étoile, cell–cell communication, likely mediated by novel transmembrane proteins that share similarities with metazoan Notch receptors, was shown to account for the establishment of filament patterning and cell differentiation in the filamentous brown alga Ectocarpus siliculosus. Brown algae are multicellular marine organisms evolutionarily distant from both metazoans and land plants. The molecular or cellular mechanisms that govern the developmental patterning in brown algae are poorly characterized. Here, we report the first morphogenetic mutant, étoile (etl), produced in the brown algal model Ectocarpus siliculosus. Genetic, cellular, and morphometric analyses showed that a single recessive locus, ETL, regulates cell differentiation: etl cells display thickening of the extracellular matrix (ECM), and the elongated, apical, and actively dividing E cells are underrepresented. As a result of this defect, the overrepresentation of round, branch-initiating R cells in the etl mutant leads to the rapid induction of the branching process at the expense of the uniaxial growth in the primary filament. Computational modeling allowed the simulation of the etl mutant phenotype by including a modified response to the neighborhood information in the division rules used to specify wild-type development. Microarray experiments supported the hypothesis of a defect in cell–cell communication, as primarily Lin-Notch-domain transmembrane proteins, which share similarities with metazoan Notch proteins involved in binary cell differentiation were repressed in etl. Thus, our study highlights the role of the ECM and of novel transmembrane proteins in cell–cell communication during the establishment of the developmental pattern in this brown alga.

EARLY DEVELOPMENT PATTERN OF THE BROWN ALGA ECTOCARPUS SILICULOSUS (ECTOCARPALES, PHAEOPHYCEAE) SPOROPHYTE1

The distant phylogenetic position of brown macroalgae from the other multicellular phyla offers the opportunity to study novel and alternative developmental processes involved in the establishment of multicellularity. At present, however, very little information is available about developmental patterning in this group. Ectocarpus siliculosus (Dillwyn) Lyngb. has uniseriate filaments and displays one of the simplest architectures in the Phaeophyceae. The aim of this study was to decipher the morphogenetic steps that lead to the development of the Ectocarpus sporophyte. We carried out a detailed morphometric study of the events that occurred between gamete germination and the 100‐cell stage. This analysis was performed on two ecologically distant isolates to assess plasticity in developmental patterning within this species. Cell sizes were measured in both isolates, allowing the definition of two main cell types based on their shape (round and elongated). On average, the filament is composed of about 40% round cells, which are present in the central region of the filament, but different combinations of the two cell types within filaments were observed and quantified. Young sporophytes grew apically, with elongated cells progressively differentiating into round cells. Secondary filaments emerged preferentially on round cells, primarily from the older central cells. Statistical analyses showed that the pattern of branching was regulated to ensure a stereotyped architecture. This description of the developmental patterning during the growth of the E. siliculosus sporophyte will serve as a base for more detailed studies of development, in this species and in brown algae in general.

A stochastic 1D nearest-neighbour automaton models early development of the brown alga Ectocarpus siliculosus

Early development of the filamentous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye involves two cell types that are arranged in a polymorphic, but constrained, pattern. The present study aimed to decipher the cellular processes responsible for the establishment of this pattern. Thorough observations characterised five different events of division and differentiation that occurred during the early development. The hypothesis that a local control is responsible for these processes was tested. To do so, Ectomat, a stochastic automaton in which each cell only interacts with its closest neighbour(s), was created. The probabilities for the five events were adjusted to fit to the observations. Simulations with Ectomat reconstructed most of the essential properties of the sporophyte development, in terms of cell-type proportion, relative position and growth dynamics. The whole organism properties emerged by applying local transition rules. In conclusion, no global position information system was required at this development stage. Randomly occurring cell events, driven by simple contact interactions, are sufficient to account for the early filament development and establishment of the cell-type pattern of E. siliculosus.

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.

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.

Space-time decoupling in the branching process in the mutant étoile of the filamentous brown alga Ectocarpus siliculosus

Ectocarpus siliculosus is being developed as a model organism for brown algal genetics and genomics.1,2 Brown algae are phylogenetically distant from the other multicellular phyla (green lineage, red algae, fungi and metazoan)3 and therefore might offer the opportunity to study novel and alternative developmental processes that lead to the establishment of multicellularity. E. siliculosus develops as uniseriate filaments, thereby displaying one of the simplest architectures among multicellular organisms.4 The young sporophyte grows as a primary filament and then branching occurs, preferentially at the center of the filament. We recently described the first morphogenetic mutant étoile (etl) in a brown alga, produced by UVB mutagenesis in E. siliculosus.5 We showed that a single recessive mutation was responsible for a defect in both cell differentiation and the very early branching pattern (first and second branch emergences). Here, we supplement this study by reporting the branching defects observed subsequently, i.e. for the later stages corresponding to the emergence of up to the first six secondary filaments, and we show that the branching process is composed of at least two distinct components: time and position. The developmental pattern of E. siliculosus is characterized by a very high level of morphological plasticity.6 Observations followed by statistical analyses allowed analyzing the morphometric features accompanying the establishment of the branching pattern in the mutant étoile, compared with the wild type (WT) organism (strain Ec32). The branching pattern can be deciphered in two main components: (1) the timing of branching and (2) the position of branching.

Neurogenetic asymmetries in the catshark developing habenulae: mechanistic and evolutionary implications

Analysis of the establishment of epithalamic asymmetry in two non-conventional model organisms, a cartilaginous fish and a lamprey, has suggested that an essential role of Nodal signalling, likely to be ancestral in vertebrates, may have been largely lost in zebrafish. In order to decipher the cellular mechanisms underlying this divergence, we have characterised neurogenetic asymmetries during habenular development in the catshark Scyliorhinus canicula and addressed the mechanism involved in this process. As in zebrafish, neuronal differentiation starts earlier on the left side in the catshark habenulae, suggesting the conservation of a temporal regulation of neurogenesis. At later stages, marked, Alk4/5/7 dependent, size asymmetries having no clear counterparts in zebrafish also develop in neural progenitor territories, with a larger size of the proliferative, pseudostratified neuroepithelium, in the right habenula relative to the left one, but a higher cell number on the left of a more lateral, later formed population of neural progenitors. These data show that mechanisms resulting in an asymmetric, preferential maintenance of neural progenitors act both in the left and the right habenulae, on different cell populations. Such mechanisms may provide a substrate for quantitative variations accounting for the variability in size and laterality of habenular asymmetries across vertebrates.