Stepwise emergence of the neuronal gene expression program in early animal evolution.

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.

The concept of intrinsic versus extrinsic apoptosis.

Regulated cell death is a vital and dynamic process in multicellular organisms that maintains tissue homeostasis and eliminates potentially dangerous cells. Apoptosis, one of the better-known forms of regulated cell death, is activated when cell-surface death receptors like Fas are engaged by their ligands (the extrinsic pathway) or when BCL-2-family pro-apoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both the intrinsic and extrinsic pathways of apoptosis lead to the activation of a family of proteases, the caspases, which are responsible for the final cell demise in the so-called execution phase of apoptosis. In this review, I will first discuss the most common types of regulated cell death on a morphological basis. I will then consider in detail the molecular pathways of intrinsic and extrinsic apoptosis, discussing how they are activated in response to specific stimuli and are sometimes overlapping. In-depth knowledge of the cellular mechanisms of apoptosis is becoming more and more important not only in the field of cellular and molecular biology but also for its translational potential in several pathologies, including neurodegeneration and cancer.

The genetic program for cartilage development has deep homology within Bilateria.

The evolution of novel cell types led to the emergence of new tissues and organs during the diversification of animals. The origin of the chondrocyte, the cell type that synthesizes cartilage matrix, was central to the evolution of the vertebrate endoskeleton. Cartilage-like tissues also exist outside the vertebrates, although their relationship to vertebrate cartilage is enigmatic. Here we show that protostome and deuterostome cartilage share structural and chemical properties, and that the mechanisms of cartilage development are extensively conserved--from induction of chondroprogenitor cells by Hedgehog and ß-catenin signalling, to chondrocyte differentiation and matrix synthesis by SoxE and SoxD regulation of clade A fibrillar collagen (ColA) genes--suggesting that the chondrogenic gene regulatory network evolved in the common ancestor of Bilateria. These results reveal deep homology of the genetic program for cartilage development in Bilateria and suggest that activation of this ancient core chondrogenic network underlies the parallel evolution of cartilage tissues in Ecdysozoa, Lophotrochozoa and Deuterostomia.

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes.

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.

BioCell2XML: a novel tool for converting cell lineage data from SIMI BioCell to MaMuT (Fiji).

Computer-assisted 4D manual cell tracking has been a valuable method for understanding spatial-temporal dynamics of embryogenesis (e.g., Stach & Anselmi BMC Biol, 13(113), 1-11 2015; Vellutini et al. BMC Biol, 15(33), 1-28 2017; Wolff et al. eLife, 7, e34410 2018) since the method was introduced in the late 1990s. Since two decades SIMI® BioCell (Schnabel et al. Dev Biol, 184, 234-265 1997), a software which initially was developed for analyzing data coming from the, at that time new technique of 4D microscopy, is in use. Many laboratories around the world use SIMI BioCell for the manual tracing of cells in embryonic development of various species to reconstruct cell genealogies with high precision. However, the software has several disadvantages: limits in handling very large data sets, the virtually no maintenance over the last 10 years (bound to older Windows versions), the difficulty to access the created cell lineage data for analyses outside SIMI BioCell, and the high cost of the program. Recently, bioinformatics, in close collaboration with biologists, developed new lineaging tools that are freely available through the open source image processing platform Fiji. Here we introduce a software tool that allows conversion of SIMI BioCell lineage data to a format that is compatible with the Fiji plugin MaMuT (Wolff et al. eLife, 7, e34410 2018). Hereby we intend to maintain the usability of SIMI BioCell created cell lineage data for the future and, for investigators who wish to do so, facilitate the transition from this software to a more convenient program.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Presynaptic active zones in invertebrates and vertebrates.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

Biomonitoring of water quality of the Osumi, Devolli, and Shkumbini rivers through benthic macroinvertebrates and chemical parameters.

Environmental monitoring of river water quality in Albania, using biological and chemical parameters, is a fast and effective way to assess the quality of water bodies.The aim of this study was to investigate Ephemeroptera, Plecoptera and Trichoptera (EPT), Biotic index-Richness using macroinvertebrates to assess the water quality, with special reference to nutrient (phosphorus and nitrogen) levels in the Devolli, Shkumbini and Osumi rivers. Our objective was to investigate the relationships between the measures of benthic macroinvertebrate communities and nutrient concentrations to assess water quality. The rivers' benthic macroinvertebrates were collected during different seasons in 2012. The biological and chemical parameters used in the current study identified them as quick indicators of water quality assessment. The total number of macroinvertebrate individuals (n = 15,006) (Osumi river: n = 5,546 organisms; Devolli river: n = 3,469 organisms; and Shkumbini river: n = 5,991 organisms), together with the EPT group (Ephemeroptera, Plecoptera, and Trichoptera), showed that the water quality at the river stations during the above-mentioned period belonged to Classes II and III (fair water quality and good water quality, respectively). The classification of the water quality was also based on the nitrogen and total phosphorus contents. The pollution tolerance levels of macroinvertebrate taxa varied from the non-tolerating forms encountered in environments with low pollution levels to the tolerating forms that are typical of environments with considerable pollution levels.

Oocyte aging in a marine protostome worm: The roles of maturation-promoting factor and extracellular signal regulated kinase form of mitogen-activated protein kinase.

The roles of maturation-promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen-activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase-I-arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho-specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation-initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.

The study of Priapulus caudatus reveals conserved molecular patterning underlying different gut morphogenesis in the Ecdysozoa.

BACKGROUND: The digestive systems of animals can become highly specialized in response to their exploration and occupation of new ecological niches. Although studies on different animals have revealed commonalities in gut formation, the model systems Caenorhabditis elegans and Drosophila melanogaster, which belong to the invertebrate group Ecdysozoa, exhibit remarkable deviations in how their intestines develop. Their morphological and developmental idiosyncrasies have hindered reconstructions of ancestral gut characters for the Ecdysozoa, and limit comparisons with vertebrate models. In this respect, the phylogenetic position, and slow evolving morphological and molecular characters of marine priapulid worms advance them as a key group to decipher evolutionary events that occurred in the lineages leading to C. elegans and D. melanogaster. RESULTS: In the priapulid Priapulus caudatus, the gut consists of an ectodermal foregut and anus, and a mid region of at least partial endodermal origin. The inner gut develops into a 16-cell primordium devoid of visceral musculature, arranged in three mid tetrads and two posterior duplets. The mouth invaginates ventrally and shifts to a terminal anterior position as the ventral anterior ectoderm differentially proliferates. Contraction of the musculature occurs as the head region retracts into the trunk and resolves the definitive larval body plan. Despite obvious developmental differences with C. elegans and D. melanogaster, the expression in P. caudatus of the gut-related candidate genes NK2.1, foxQ2, FGF8/17/18, GATA456, HNF4, wnt1, and evx demonstrate three distinct evolutionarily conserved molecular profiles that correlate with morphologically identified sub-regions of the gut. CONCLUSIONS: The comparative analysis of priapulid development suggests that a midgut formed by a single endodermal population of vegetal cells, a ventral mouth, and the blastoporal origin of the anus are ancestral features in the Ecdysozoa. Our molecular data on P. caudatus reveal a conserved ecdysozoan gut-patterning program and demonstrates that extreme morphological divergence has not been accompanied by major molecular innovations in transcriptional regulators during digestive system evolution in the Ecdysozoa. Our data help us understand the origins of the ecdysozoan body plan, including those of C. elegans and D. melanogaster, and this is critical for comparisons between these two prominent model systems and their vertebrate counterparts.

Two new species of dicyemid mesozoans (Dicyemida: Dicyemidae) from Octopus maya Voss & Solis-Ramirez (Octopodidae) off Yucatan, Mexico.

Two new dicyemid species are described from the endemic cephalopod Octopus maya Voss & Solis-Ramirez collected off Yucatan, Mexico. The renal sacs of 40 juvenile and adult octopuses from four localities were examined. Dicyema hochbergi n. sp. is a medium-sized species that reaches 2,245 µm in length. The vermiform stages consist of 18-24 peripheral cells, a conical calotte and the extension of the axial cell between the base and middle of the metapolar cells. Infusoriform embryos consist of 39 cells with urn cell containing one germinal cell, two nuclei and solid refringent bodies. Dicyema mexcayae n. sp. is a relatively small species that reaches 1,114 µm in length. The vermiform stages are constituted by 14-16 peripheral cells, an elongate calotte and the axial cell extending forward to the middle of the metapolar cells. The infusoriform embryos consist of 37 cells, two solid refringent bodies and urn cells with two nuclei each. The present study represents the first description of a dicyemid species from O. maya and increases the number of described species from Mexican waters to 11.

Metamorphic remodeling of morphology and the body cavity in Phoronopsis harmeri (Lophotrochozoa, Phoronida): the evolution of the phoronid body plan and life cycle.

BACKGROUND: Phoronids undergo a remarkable metamorphosis, in which some parts of the larval body are consumed by the juvenile and the body plan completely changes. According to the only previous hypothesis concerning the evolution of the phoronid body plan, a hypothetical ancestor of phoronids inhabited a U-shaped burrow in soft sediment, where it drew the anterior and posterior parts of the body together and eventually fused them. In the current study, we investigated the metamorphosis of Phoronopsis harmeri with light, electron, and laser confocal microscopy. RESULTS: During metamorphosis, the larval hood is engulfed by the juvenile; the epidermis of the postroral ciliated band is squeezed from the tentacular epidermis and then engulfed; the larval telotroch undergoes cell death and disappears; and the juvenile body forms from the metasomal sack of the larva. The dorsal side of the larva becomes very short, whereas the ventral side becomes very long. The terminal portion of the juvenile body is the ampulla, which can repeatedly increase and decrease in diameter. This flexibility of the ampulla enables the juvenile to dig into the sediment. The large blastocoel of the larval collar gives rise to the lophophoral blood vessels of the juvenile. The dorsal blood vessel of the larva becomes the definitive median blood vessel. The juvenile inherits the larval protocoel, mesocoel, and metacoel. Late in metamorphosis, however, the protocoel loses its epithelial structure: the desmosomes between cells and the basal lamina under the cells disappear. This loss may reflect a reduction of the protocoel, which is a characteristic of some recent phoronids. CONCLUSIONS: Based on our investigation of P. harmeri metamorphosis, we hypothesize that the phoronid ancestor was worm-like animal that possessed preoral, tentacular, and trunk coeloms. It lived on the soft sediment and collected food with its tentacles. When threatened, this worm-like ancestor buried itself in the soft sediment by means of the ventral protrusion into which the loop of the intestine and the blood vessels were drawn. We propose that this behavior gave rise to the body plan of all recent phoronids. The evolution of phoronid life cycle seems having more in common with"intercalation" than "terminal addition" theories.

Calcium signaling and endoplasmic reticulum dynamics during fertilization in marine protostome worms belonging to the phylum Nemertea.

Metaphase-I-arrested eggs of marine protostome worms in the phylum Nemertea generate a series of point-source calcium waves during fertilization. Such calcium oscillations depend on inositol-1,4,5-trisphosphate-mediated calcium release from endoplasmic reticulum (ER) stores that undergo structural reorganizations prior to and after fertilization. This article reviews fertilization-induced calcium transients and ER dynamics in nemertean eggs and compares these topics to what has been reported for other animals in order to identify unifying characteristics and distinguishing features of calcium responses during fertilization across the animal kingdom.

[Transcriptional control of ciliary genes]. / Contrôle transcriptionnel des gènes ciliaires.

Cilia are found in many eukaryotic species and share a common microtubule architecture that can nonetheless show very diverse features within one animal. The genesis of cilia and their diversity require the expression of different specific genes. At least two classes of transcription factors are involved in ciliogenesis: the RFX family, essential for the assembly of most cilia and the FOXJ1 transcription factors that are key regulators of motile cilia assembly. These two different families of transcription factors have both specific and common target genes and they can also cooperate for the formation of cilia. In collaboration with cell type specific factors, they also contribute to the specialisation of cilia. As a consequence, the identification of RFX and FOXJ1 target genes has emerged as an efficient strategy to identify novel ciliary genes, and in particular genes potentially implicated in ciliopathies.

Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments.

Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies.

Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features.

BACKGROUND: Inferences concerning the evolution of invertebrate nervous systems are often hampered by the lack of a solid data base for little known but phylogenetically crucial taxa. In order to contribute to the discussion concerning the ancestral neural pattern of the Lophotrochozoa (a major clade that includes a number of phyla that exhibit a ciliated larva in their life cycle), we investigated neurogenesis in Phoronopsis harmeri, a member of the poorly studied Phoronida, by using antibody staining against serotonin and FMRFamide in combination with confocal microscopy and 3D reconstruction software. RESULTS: The larva of Phoronopsis harmeri exhibits a highly complex nervous system, including an apical organ that consists of four different neural cell types, such as numerous serotonin-like immunoreactive flask-shaped cells. In addition, serotonin- and FMRFamide-like immunoreactive bi- or multipolar perikarya that give rise to a tentacular neurite bundle which innervates the postoral ciliated band are found. The preoral ciliated band is innervated by marginal serotonin-like as well as FMRFamide-like immunoreactive neurite bundles. The telotroch is innervated by two neurite bundles. The oral field is the most densely innervated area and contains ventral and ventro-lateral neurite bundles as well as several groups of perikarya. The digestive system is innervated by both serotonin- and FMRFamide-like immunoreactive neurites and perikarya. Importantly, older larvae of P. harmeri show a paired ventral neurite bundle with serial commissures and perikarya. CONCLUSIONS: Serotonin-like flask-shaped cells such as the ones described herein for Phoronopsis harmeri are found in the majority of lophotrochozoan larvae and therefore most likely belong to the ground pattern of the last common lophotrochozoan ancestor. The finding of a transitory paired ventral neurite bundle with serially repeated commissures that disappears during metamorphosis suggests that such a structure was part of the "ur-phoronid" nervous system, but was lost in the adult stage, probably due to its acquired sessile benthic lifestyle.

Ventral nerve cord in Phoronopsis harmeri larvae.

The nervous system organization is considered a phylogenetically important character among metazoans. The phylum Phoronida is included in a supraphyletic taxon known as Lophotrochozoa. Many lophotrochozoans possess a metameric ventral nerve cord as adults or larvae. Phoronids do not exhibit external metamery either as larvae or as adults. The current study describes the ventral nerve cord in the young larva of Phoronopsis harmeri. This structure is apparent both in the serotonergic and FMRF-amidergic nervous system in young larvae. The ventral nerve cord extends from the mouth to the tentacular ridge. Both serotonergic and FMRF-amidergic components consist of two ventrolateral nerves, each with several unipolar neurons. The ventrolateral nerves connect to each other by means of thin repetitive transversal nerves ("commissures"). The abundance of neurons and nerves in the epidermis of the oral field of actinotrocha larva likely reflects the importance of this area in collection of food particles. The ventral nerve cords of the actinotrocha and the metatrochophore differ in their positions with respect to ciliated bands: the cord is located between the preoral and postoral ciliated bands in the actinotrocha but between the postoral ciliated band and telotroch in the metatrochophore. The presence of the ventral nerve cord, which contains repetitive elements (neurons and "commissures"), in the early development of P. harmeri may recapitulate some stages of nervous system development during phoronid phylogeny. The larval nervous system does not contain nervous centers under the tentacular ridge that can correlate with the catastrophic metamorphosis and unique body plan of phoronids.

Segmental mode of neural patterning in sipuncula.

Recent molecular phylogenetic analyses suggest a close relationship between two worm-shaped phyla, the nonsegmented Sipuncula (peanut worms) and the segmented Annelida (e.g., earthworms and polychaetes) [1-5]. The striking differences in their bodyplans are exemplified by the annelids' paired, ladder-like ventral nervous system, which contains segmentally arranged ganglia, and the sipunculans' single ventral nerve cord (VNC), which is devoid of any segmental structures [6, 7]. Investigating central nervous system (CNS) formation with serotonin and FMRFamide labeling in a representative sipunculan, Phascolosoma agassizii, we found that neurogenesis initially follows a segmental pattern similar to that of annelids. Starting out with paired FMRFamidergic and serotonergic axons, four pairs of associated serotonergic perikarya and interconnecting commissures form one after another in an anterior-posterior progression. In late-stage larvae, the two serotonergic axons of the VNCs fuse, the commissures disappear, and one additional pair of perikarya is formed. These cells (ten in total) migrate toward one another, eventually forming two clusters of five cells each. These neural-remodeling processes result in the single nonmetameric CNS of the adult sipunculan. Our data confirm the segmental ancestry of Sipuncula and render Phascolosoma a textbook example for the Haeckelian hypothesis of ontogenetic recapitulation of the evolutionary history of a species [8].

Divide and conquer: how asymmetric division shapes cell fate in the hematopoietic system.

A fundamental mechanism by which cells can give rise to daughters with different fates is via asymmetric division. During asymmetric division, a mother cell generates daughter cells that go on to adopt different fates because of differential segregation of cell fate determinants. Although originally characterized in invertebrates, asymmetric division has recently been shown to regulate cell fate decisions in the mammalian hematopoietic system, playing crucial roles in stem cell renewal, lymphocyte activation, and leukemogenesis. These discoveries have opened new doors to understanding how regulation of division pattern contributes to the normal development and function of the immune system as well as how its dysregulation can lead to cancer.