Lateral cell polarization drives organization of epithelia in sea anemone embryos and embryonic cell aggregates.

One of the first organizing processes during animal development is the assembly of embryonic cells into epithelia. In certain animals, including Hydra and sea anemones, epithelia also emerge when cells from dissociated tissues are aggregated back together. Although cell adhesion is required to keep cells together, it is not clear whether cell polarization plays a role as epithelia emerge from disordered aggregates. Here, we demonstrate that lateral cell polarization is essential for epithelial organization in both embryos and aggregates of the sea anemone Nematostella vectensis. Specifically, knock down of the lateral polarity protein Lgl disrupts epithelia in developing embryos and impairs the capacity of dissociated cells to epithelialize from aggregates. Cells in lgl mutant epithelia lose their columnar shape and have mispositioned mitotic spindles and ciliary basal bodies. Together, our data suggest that in Nematostella, Lgl is required to establish lateral cell polarity and position cytoskeletal organelles in cells of embryos and aggregates during de novo epithelial organization.

Origin and development of primary animal epithelia.

Epithelia are the first organized tissues that appear during development. In many animal embryos, early divisions give rise to a polarized monolayer, the primary epithelium, rather than a random aggregate of cells. Here, we review the mechanisms by which cells organize into primary epithelia in various developmental contexts. We discuss how cells acquire polarity while undergoing early divisions. We describe cases where oriented divisions constrain cell arrangement to monolayers including organization on top of yolk surfaces. We finally discuss how epithelia emerge in embryos from animals that branched early during evolution and provide examples of epithelia-like arrangements encountered in single-celled eukaryotes. Although divergent and context-dependent mechanisms give rise to primary epithelia, here we trace the unifying principles underlying their formation.

Topological structures and syntenic conservation in sea anemone genomes.

There is currently little information about the evolution of gene clusters, genome architectures and karyotypes in early branching animals. Slowly evolving anthozoan cnidarians can be particularly informative about the evolution of these genome features. Here we report chromosome-level genome assemblies of two related anthozoans, the sea anemones Nematostella vectensis and Scolanthus callimorphus. We find a robust set of 15 chromosomes with a clear one-to-one correspondence between the two species. Both genomes show chromosomal conservation, allowing us to reconstruct ancestral cnidarian and metazoan chromosomal blocks, consisting of at least 19 and 16 ancestral linkage groups, respectively. We show that, in contrast to Bilateria, the Hox and NK clusters of investigated cnidarians are largely disintegrated, despite the presence of staggered hox/gbx expression in Nematostella. This loss of microsynteny conservation may be facilitated by shorter distances between cis-regulatory sequences and their cognate transcriptional start sites. We find no clear evidence for topologically associated domains, suggesting fundamental differences in long-range gene regulation compared to vertebrates. These data suggest that large sets of ancestral metazoan genes have been retained in ancestral linkage groups of some extant lineages; yet, higher order gene regulation with associated 3D architecture may have evolved only after the cnidarian-bilaterian split.

Cell polarity oscillations in mitotic epithelia.

Epithelial organization and function depend on coordinated cell polarity. In developing tissues, proliferative epithelia maintain whole tissue polarity as individual cells undergo symmetric divisions. However, recent work has shown that cells in diverse epithelia remodel their polarity in a cell cycle-dependent manner. Here, we discuss the different mechanisms that drive mitotic polarity oscillations and their implications for tissue morphogenesis.

Cell-Cycle-Coupled Oscillations in Apical Polarity and Intercellular Contact Maintain Order in Embryonic Epithelia.

Throughout animals, embryonic cells must ultimately organize into polarized epithelial layers that provide the structural basis for gastrulation or subsequent developmental events [1]. Precisely how this primary epithelium maintains continuous integrity during rapid and repeated cell divisions has never been directly addressed, particularly in cases where early cleavages are driven in synchrony. Representing the early-branching non-bilaterian phylum Cnidaria, embryos of the sea anemone Nematostella vectensis undergo rapid synchronous cell divisions and ultimately give rise to a diploblastic epithelial body plan after gastrulation [2, 3]. Here, using live imaging of apical polarity proteins in Nematostella embryos, we demonstrate that cell polarity is established by the four-cell stage and then reiteratively lost during subsequent mitoses, correlating with transient adhesion disengagement and dramatic deformations of embryonic morphology. Intriguingly, the re-establishment of polarity and adhesion during each interphase is associated with a process of whole-embryo compaction analogous to that observed in mammals [4-7]. Because similar protein dynamics are observed in dividing epithelial cells in Drosophila melanogaster, we propose that cell-cycle-coupled oscillations in apical polarity may be conserved throughout Metazoa.

Cell division and the maintenance of epithelial order.

Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis.

A single GATA factor plays discrete, lineage specific roles in ascidian heart development.

GATA family transcription factors are core components of the vertebrate heart gene network. GATA factors also contribute to heart formation indirectly through regulation of endoderm morphogenesis. However, the precise impact of GATA factors on vertebrate cardiogenesis is masked by functional redundancy within multiple lineages. Early heart specification in the invertebrate chordate Ciona intestinalis is similar to that of vertebrates but only one GATA factor, Ci-GATAa, is expressed in the heart progenitor cells and adjacent endoderm. Here we delineate precise, tissue specific contributions of GATAa to heart formation. Targeted repression of GATAa activity in the heart progenitors perturbs their transcriptional identity. Targeted repression of endodermal GATAa function disrupts endoderm morphogenesis. Subsequently, the bilateral heart progenitors fail to fuse at the ventral midline. The resulting phenotype is strikingly similar to cardia bifida, as observed in vertebrate embryos when endoderm morphogenesis is disturbed. These findings indicate that GATAa recapitulates cell-autonomous and non-cell-autonomous roles performed by multiple, redundant GATA factors in vertebrate cardiogenesis.

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor.

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.

The Bacillus subtilis spore coat provides "eat resistance" during phagocytic predation by the protozoan Tetrahymena thermophila.

Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple "extracellular" layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes.

Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores.

The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat. GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat. Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase. GerQ is the only substrate for this transglutaminase identified to date. In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis. These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.

Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate.

Bacillus subtilis spores can germinate with a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca(2+)-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca(2+)-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is sigma(E) dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ.