Choanoflagellates, choanocytes, and animal multicellularity

Abstract. It is widely accepted that multicellular animals (metazoans) constitute a monophyletic unit, deriving from ancestral choanoflagellate‐like protists that gave rise to simple choanocyte‐bearing metazoans. However, a re‐assessment of molecular and histological evidence on choanoflagellates, sponge choanocytes, and other metazoan cells reveals that the status of choanocytes as a fundamental cell type in metazoan evolution is unrealistic. Rather, choanocytes are specialized cells that develop from non‐collared ciliated cells during sponge embryogenesis. Although choanocytes of adult sponges have no obvious homologue among metazoans, larval cells transdifferentiating into choanocytes at metamorphosis do have such homologues. The evidence reviewed here also indicates that sponge larvae are architecturally closer than adult sponges to the remaining metazoans. This may mean that the basic multicellular organismal architecture from which diploblasts evolved, that is, the putative planktonic archimetazoan, was more similar to a modern poriferan larva lacking choanocytes than to an adult sponge. Alternatively, it may mean that other metazoans evolved from a neotenous larva of ancient sponges. Indeed, the Porifera possess some features of intriguing evolutionary significance: (1) widespread occurrence of internal fertilization and a notable diversity of gastrulation modes, (2) dispersal through architecturally complex lecithotrophic larvae, in which an ephemeral archenteron (in dispherula larvae) and multiciliated and syncytial cells (in trichimella larvae) occur, (3) acquisition of direct development by some groups, and (4) replacement of choanocyte‐based filter‐feeding by carnivory in some sponges. Together, these features strongly suggest that the Porifera may have a longer and more complicated evolutionary history than traditionally assumed, and also that the simple anatomy of modern adult sponges may have resulted from a secondary simplification. This makes the idea of a neotenous evolution less likely than that of a larva‐like choanocyte‐lacking archimetazoan. From this perspective, the view that choanoflagellates may be simplified sponge‐derived metazoans, rather than protists, emerges as a viable alternative hypothesis. This idea neither conflicts with the available evidence nor can be disproved by it, and must be specifically re‐examined by further approaches combining morphological and molecular information. Interestingly, several microbial lin°Cages lacking choanocyte‐like morphology, such as Corallochytrea, Cristidiscoidea, Ministeriida, and Mesomycetozoea, have recently been placed at the boundary between fungi and animals, becoming a promising source of information in addition to the choanoflagellates in the search for the unicellular origin of animal multicellularity.     

A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians

The conserved family of Wnt signaling molecules mediates various developmental processes including governing cell fate, proliferation, and polarity. The diverse developmental functions of the Wnt genes in bilaterians have obscured the evolutionary origin of this important signaling pathway. Recent work in the Cnidaria has shown the diversity of Wnt genes, and regulatory components of Wnt signaling, evolved early in metazoan evolution, prior to the divergence of cnidarians and bilaterians. Evidence from Hydra and the sea anemone, Nematostella, demonstrates a role for Wnt signaling in axis formation and patterning, as well as gastrulation and germ-layer specification. In this review, we examine what is currently known about Wnt signaling in cnidarians, and discuss what this group of "simple" animals may reveal about the evolution of Wnt signaling and polarity.     

Sponge proteins are more similar to those of Homo sapiens than to Caenorhabditis elegans

We compared 42 phylogenetically conserved proteins from four marine sponges [Porifera] with almost the complete set of Caenorhabditis elegans proteins and all known proteins from humans. The majority of the sponge proteins arc significantly more similar to human than to C. elegans orthologucs/homologues. This finding reflects the accelerated evolutionary rate in the C. elegans lineage, since sponges split off first from the common ancestor of all multicellular animals. Furthermore, three sponge/human proteins were not found in C. elegans: (2–5)A synthetase, DNA repair helicase and lens βγ-crystallin. Sponges arc the source of the most ancient proteins already present in the common ancestor of all multicellular organisms. Some of these proteins were lost later during the evolution of individual animal lineages. These 'found/lost' proteins may serve as molecular markers for an improved systematics of Metazoa. In addition, phylogenetically conserved sponge proteins can be very helpful for the evaluation of differences in evolutionary rates in different animal lineages. We therefore propose sponges as the reference animals in molecular evolutionary studies of Metazoa.     

Effects of dissolved oxygen levels on survival and growth in vitro of <Emphasis Type="Italic">Haliclona pigmentifera</Emphasis> (Demospongiae)

In vitro sponge cultures are considered as legitimate alternatives for utilizing marine sponges (Porifera) to yield bioactive molecules. Optimization of culture methodologies for enhancing sponge survival is in progress for the identification of the factors regulating sponge survival in vitro. Dissolved oxygen (DO) is an essential factor promoting sponge survival. However, the effects of variable DO levels on the in vitro survival responses of sponges are not fully understood. Hence, we have investigated the effects of variable DO levels on the survival of the marine sponge, Haliclona pigmentifera (Demospongiae), with no external nutritional supplementation in closed type incubator chambers. Our results indicate that, under hypoxic conditions (1.5-2.0 ppm DO), H. pigmentifera with intact ectodermal layers and subtle oscula show adherent growth for 42±3 days. Sponges with prominent oscula, foreign material, and damaged pinacoderm exhibit poor survival under similar conditions. Complete mortality occurs within 2 days under anoxia (<0.3 ppm DO), and survival for a few days has been observed at >4.0 ppm DO without adhesion. Cellular differences between the outer and inner zones and collagen-like extracellular matrix have been identified in adherent sponges. Based on the hypothesis that hypoxia-inducible factor1-α (HIF-1α) is a ubiquitous protein promoting hypoxic survival in animals, we have detected, by Western blot, a protein band corresponding to human HIF-1α-like protein from sponges exposed to hypoxia and to hypoxia-mimicking agents. We thus report, for the first time, adhesive growth and a protein band corresponding to human HIF-1α-like protein in sponges surviving hypoxia in vitro. This work was supported by the “Task Force Network Programme (CMM-004)” of the Council of Scientific and Industrial Research (CSIR), Government of India and by the Department of Ocean Development. CSIR is also acknowledged for providing a Senior Research Fellowship to V.G.G.     

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.     

Environmental Flow Regimes for <Emphasis Type="Italic">Dysidea avara</Emphasis> Sponges

The aim of our research is to design tank systems to culture Dysidea avara for the production of avarol. Flow information was needed to design culture tanks suitable for effective production. Water flow regimes were characterized over a 1-year period for a shallow rocky sublittoral environment in the Northwestern Mediterranean where D. avara sponges are particularly abundant. Three-dimensional Doppler current velocities at 8-10-m depths ranged from 5 to 15 cm/s over most seasons, occasionally spiking to 30-66 cm/s. A thermistor flow sensor was used to map flow fields in close proximity ( approximately 2 cm) to individual sponges at 4.5-, 8.8-, and 14.3-m depths. These "proximal flows" averaged 1.6 cm/s in calm seas and 5.9 cm/s during a storm, when the highest proximal flow (32.9 cm/s) was recorded next to a sponge at the shallowest station. Proximal flows diminished exponentially with depth, averaging 2.6 cm/s +/- 0.15 SE over the entire study. Flow visualization studies showed that oscillatory flow (0.20-0.33 Hz) was the most common regime around individual sponges. Sponges at the 4.5-m site maintained a compact morphology with large oscula year-around despite only seasonally high flows. Sponges at 8.8 m were more erect with large oscula on tall protuberances. At the lowest-flow 14.3-m site, sponges were more branched and heavily conulated, with small oscula. The relationship between sponge morphology and ambient flow regime is discussed.     

贵州寒武纪海绵动物化石组合特征

贵州寒武纪海绵化石丰富。分布广泛,主要产于早寒武世梅树村期的戈仲伍组或牛蹄塘组底部、筇竹寺期的牛蹄塘组和早、中寒武世凯里组。梅树村期的海绵属种单调,主要为六射海绵类麦粒海绵（Triticispongia sp．）及一些海绵骨针,代表寒武纪海绵首次爆发或辐射的前奏和先驱。牛蹄塘组海绵多达13个属,是寒武纪海绵辐射的重要窗口,以普通海绵略占优势,Leptomitus为优势属。凯里组的海绵共计7属,以普通海绵为主。贵州寒武纪海绵动物群的发展具有明显的阶段性和组成的差异性,其演变过程受到环境的制约。贵州早期海绵动物的发现和研究不仅改变了寒武纪生物多样性的认识,而且还为探讨海绵动物本身的起源、辐射提供了重要材料。     

The many roles of PTK7: a versatile regulator of cell-cell communication

PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor with functions in various processes ranging from embryonic morphogenesis to epidermal wound repair. Here, we review recent findings indicating that PTK7 is a versatile co-receptor that functions as a molecular switch in Wnt, Semaphorin/Plexin and VEGF signaling pathways. We focus in particular on the role of PTK7 in Wnt signaling, as recent data indicate that PTK7 acts as a Wnt co-receptor, which activates the planar cell polarity pathway, but inhibits canonical Wnt signaling.     

The tangled web of non-canonical Wnt signalling in neural migration

In all multicellular animals, successful embryogenesis is dependent on the ability of cells to detect the status of the local environment and respond appropriately. The nature of the extracellular environment is communicated to the intracellular compartment by ligand/receptor interactions at the cell surface. The Wnt canonical and non-canonical signalling pathways are found in the most primitive metazoans, and they play an essential role in the most fundamental developmental processes in all multicellular organisms. Vertebrates have expanded the number of Wnts and Frizzled receptors and have additionally evolved novel Wnt receptor families (Ryk, Ror). The multiplicity of potential interactions between Wnts, their receptors and downstream effectors has exponentially increased the complexity of the signal transduction network. Signalling through each of the Wnt pathways, as well as crosstalk between them, plays a critical role in the establishment of the complex architecture of the vertebrate central nervous system. In this review, we explore the signalling networks triggered by non-canonical Wnt/receptor interactions, focussing on the emerging roles of the non-conventional Wnt receptors Ryk and Ror. We describe the role of these pathways in neural tube formation and axon guidance where Wnt signalling controls tissue polarity, coordinated cell migration and axon guidance via remodelling of the cytoskeleton.     

Multiple Protein Tyrosine Phosphatases in Sponges and Explosive Gene Duplication in the Early Evolution of Animals Before the Parazoan–Eumetazoan Split

Protein tyrosine phosphatases (PTPs) regulate various physiological events in animal cells. They comprise a diverse family which are classified into two categories, receptor type and nonreceptor type. From the domain organization and phylogenetic tree, we have classified known PTPs into 17 subtypes (9 receptor-type and 8 nonreceptor-type PTPs) which are characterized by different organization of functional domain and independent cluster in tree. The receptor type PTPs are thought to be implicated in cell–cell adhesion by association of cell adhesion molecules. Since sponges are the most primitive multicellular animals and are thought to be lacking cell cohesiveness and coordination typical of eumetazoans, cloning and sequencing of PTP cDNAs of Ephydatia fluviatilis (freshwater sponge) have been conducted by RT-PCR to determine whether or not sponges have PTP genes in their genomes. We have isolated nine PTPs, of which five are possibly receptor type. A phylogenetic tree including the sponge PTPs revealed that most of the gene duplications that gave rise to the 17 subtypes had been completed in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among extant animal phyla. The family tree also revealed the rapid evolutionary rate of PTP subtypes in the early stage of animal evolution. Received: 22 October 1998 / Accepted: 27 November 1998     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Pyrvinium, a potent small molecule Wnt inhibitor, promotes wound repair and post-MI cardiac remodeling

Wnt signaling plays an important role in developmental and stem cell biology. To test the hypothesis that temporary inhibition of Wnt signaling will enhance granulation tissue and promote angiogenesis in tissue repair, we employed a recently characterized small molecule Wnt inhibitor. Pyrvinium is an FDA-approved drug that we identified as a Wnt inhibitor in a chemical screen for small molecules that stabilize β-catenin and inhibit Axin degradation. Our subsequent characterization of pyrvinium has revealed that its critical cellular target in the Wnt pathway is Casein Kinase 1α. Daily administration of pyrvinium directly into polyvinyl alcohol (PVA) sponges implanted subcutaneously in mice generated better organized and vascularized granulation tissue; this compound also increased the proliferative index of the tissue within the sponges. To evaluate its effect in myocardial repair, we induced a myocardial infarction (MI) by coronary artery ligation and administered a single intramyocardial dose of pyrvinium. Mice were evaluated by echocardiography at 7 and 30 days post-MI and treatment; post mortem hearts were evaluated by histology at 30 days. Pyrvinium reduced adverse cardiac remodeling demonstrated by decreased left ventricular internal diameter in diastole (LVIDD) as compared to a control compound. Increased Ki-67+ cells were observed in peri-infarct and distal myocardium of pyrvinium-treated animals. These results need to be further followed-up to determine if therapeutic inhibition of canonical Wnt may avert adverse remodeling after ischemic injury and its impact on myocardial repair and regeneration.     

Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless

The Frizzled (Fz) protein in Drosophila is a bifunctional receptor that acts through a GTPase pathway in planar polarity signaling and as a receptor for Wingless (Wg) using the canonical Wnt pathway. We found that the ligand-binding domain (CRD) of Fz has an approximately 10-fold lower affinity for Wg than the CRD of DFz2, a Wg receptor without polarity activity. When the Fz CRD is replaced by the high-affinity CRD of DFz2, the resulting chimeric protein gains Wg signaling activity, yet also retains polarity signaling activity. In contrast, the reciprocal exchange of the Fz CRD onto DFz2 is not sufficient to confer polarity activity to DFz2. This suggests that Fz has an intrinsic capacity for polarity signaling and that high-affinity interaction with Wg couples it to the Wnt pathway.     

A novel noncanonical Wnt pathway is involved in the regulation of the asymmetric B cell division in C. elegans

The polarities of several cells that divide asymmetrically during Caenorhabditis elegans development are controlled by Wnt signaling. LIN-44/Wnt and LIN-17/Fz control the polarities of cells in the tail of developing C. elegans larvae, including the male-specific blast cell, B, that divides asymmetrically to generate a larger anterior daughter and a smaller posterior daughter. We determined that WRM-1 and the major canonical Wnt pathway components: BAR-1, SGG-1/GSK-3 and PRY-1/Axin were not involved in the control of B cell polarity. However, POP-1/Tcf is involved and is asymmetrically distributed to the B daughter nuclei, as it is in many cell divisions during C. elegans development. Aspects of the B cell division are reminiscent of the divisions controlled by the planar cell polarity (PCP) pathway that has been described in both Drosophila and vertebrate systems. We identified C. elegans homologs of Wnt/PCP signaling components and have determined that many of them appear to be involved in the regulation of B cell polarity. Specifically, MIG-5/Dsh, RHO-1/RhoA and LET-502/ROCK appear to play major roles, while other PCP components appear to play minor roles. We conclude that a noncanonical Wnt pathway, which is different from other Wnt pathways in C. elegans, regulates B cell polarity.     

C. elegans POP-1/TCF functions in a canonical Wnt pathway that controls cell migration and in a noncanonical Wnt pathway that controls cell polarity

In Caenorhabditis elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a (beta)-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. I have found that this is the case for of a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. I have used a novel RNA-mediated interference technique to interfere specifically with pop-1 zygotic function and have determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans (beta)-catenin homologs appeared to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimicked the defects in egl-20/Wnt mutants and blocked the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities.