Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis.

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here, we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis, which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFß, Notch, and FGF signalling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed, and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella, with implications for understanding the reproductive and regenerative biology of other cnidarian species.

Ascending thoracic endovascular aortic repair with chimney stent graft to free vein coronary bypass for ruptured type A aortic dissection.

The standard treatment for ruptured type A aortic dissection is open surgical repair. We have described the case of a frail patient with home oxygen-dependent chronic obstructive pulmonary disease and prior free vein circumflex coronary artery bypass who had presented with a ruptured type A aortic dissection and was deemed too high risk for open surgery. On July 7, 2017, the patient underwent emergent endovascular ruptured ascending thoracic aortic aneurysm repair with a chimney stent graft to a free vein coronary bypass that originated from the ascending thoracic aorta. The procedure was uneventful, and the patient was discharged home on postoperative day 1.

The brain regulatory program predates central nervous system evolution.

Understanding how brains evolved is critical to determine the origin(s) of centralized nervous systems. Brains are patterned along their anteroposterior axis by stripes of gene expression that appear to be conserved, suggesting brains are homologous. However, the striped expression is also part of the deeply conserved anteroposterior axial program. An emerging hypothesis is that similarities in brain patterning are convergent, arising through the repeated co-option of axial programs. To resolve whether shared brain neuronal programs likely reflect convergence or homology, we investigated the evolution of axial programs in neurogenesis. We show that the bilaterian anteroposterior program patterns the nerve net of the cnidarian Nematostella along the oral-aboral axis arguing that anteroposterior programs regionalized developing nervous systems in the cnidarian-bilaterian common ancestor prior to the emergence of brains. This finding rejects shared patterning as sufficient evidence to support brain homology and provides functional support for the plausibility that axial programs could be co-opted if nervous systems centralized in multiple lineages.

Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis.

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis , which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFß, Notch, and FGF signaling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella , with implications for understanding the reproductive and regenerative biology of other cnidarian species.

Temperature Controls eDNA Persistence across Physicochemical Conditions in Seawater.

Environmental DNA (eDNA) quantification and sequencing are emerging techniques for assessing biodiversity in marine ecosystems. Environmental DNA can be transported by ocean currents and may remain at detectable concentrations far from its source depending on how long it persist. Thus, predicting the persistence time of eDNA is crucial to defining the spatial context of the information derived from it. To investigate the physicochemical controls of eDNA persistence, we performed degradation experiments at temperature, pH, and oxygen conditions relevant to the open ocean and the deep sea. The eDNA degradation process was best explained by a model with two phases with different decay rate constants. During the initial phase, eDNA degraded rapidly, and the rate was independent of physicochemical factors. During the second phase, eDNA degraded slowly, and the rate was strongly controlled by temperature, weakly controlled by pH, and not controlled by dissolved oxygen concentration. We demonstrate that marine eDNA can persist at quantifiable concentrations for over 2 weeks at low temperatures (≤10 °C) but for a week or less at ≥20 °C. The relationship between temperature and eDNA persistence is independent of the source species. We propose a general temperature-dependent model to predict the maximum persistence time of eDNA detectable through single-species eDNA quantification methods.

Characterization of the dynamics and variability of neuronal subtype responses during growth, degrowth, and regeneration of Nematostella vectensis.

BACKGROUND: The ability to regenerate body parts is a feature of metazoan organisms and the focus of intense research aiming to understand its basis. A number of mechanisms involved in regeneration, such as proliferation and tissue remodeling, affect whole tissues; however, little is known on how distinctively different constituent cell types respond to the dynamics of regenerating tissues. Preliminary studies suggest that a number of organisms alter neuronal numbers to scale with changes in body size. In some species with the ability of whole-body axis regeneration, it has additionally been observed that regenerates are smaller than their pre-amputated parent, but maintain the correct morphological proportionality, suggesting that scaling of tissue and neuronal numbers also occurs. However, the cell dynamics and responses of neuronal subtypes during nervous system regeneration, scaling, and whole-body axis regeneration are not well understood in any system. The cnidarian sea anemone Nematostella vectensis is capable of whole-body axis regeneration, with a number of observations suggesting the ability to alter its size in response to changes in feeding. We took advantage of Nematostella's transparent and "simple" body plan and the NvLWamide-like mCherry fluorescent reporter transgenic line to probe the response of neuron populations to variations in body size in vivo in adult animals during body scaling and regeneration. RESULTS: We utilized the previously characterized NvLWamide-like::mCherry transgenic reporter line to determine the in vivo response of neuronal subtypes during growth, degrowth, and regeneration. Nematostella alters its size in response to caloric intake, and the nervous system responds by altering neuronal number to scale as the animal changes in size. Neuronal numbers in both the endodermal and ectodermal nerve nets decreased as animals shrunk, increased as they grew, and these changes were reversible. Whole-body axis regeneration resulted in regenerates that were smaller than their pre-amputated size, and the regenerated nerve nets were reduced in neuronal number. Different neuronal subtypes had distinct responses during regeneration, including consistent, not consistent, and conditional increases in number. Conditional responses were regulated, in part, by the size of the remnant fragment and the position of the amputation site. Regenerates and adults with reduced nerve nets displayed normal behaviors, indicating that the nerve net retains functionality as it scales. CONCLUSION: These data suggest that the Nematostella nerve net is dynamic, capable of scaling with changes in body size, and that neuronal subtypes display differential regenerative responses, which we propose may be linked to the scale state of the regenerating animals.

Reverse Genetic Approaches to Investigate the Neurobiology of the Cnidarian Sea Anemone Nematostella vectensis.

The cnidarian sea anemone Nematostella vectensis has grown in popularity as a model system to complement the ongoing work in traditional bilaterian model species (e.g. Drosophila, C. elegans, vertebrate). The driving force behind developing cnidarian model systems is the potential of this group of animals to impact EvoDevo studies aimed at better determining the origin and evolution of bilaterian traits, such as centralized nervous systems. However, it is becoming apparent that cnidarians have the potential to impact our understanding of regenerative neurogenesis and systems neuroscience. Next-generation sequencing and the development of reverse genetic approaches led to functional genetics becoming routine in the Nematostella system. As a result, researchers are beginning to understand how cnidarian nerve nets are related to the bilaterian nervous systems. This chapter describes the methods for morpholino and mRNA injections to knockdown or overexpress genes of interest, respectively. Carrying out these techniques in Nematostella requires obtaining and preparing embryos for microinjection, designing and generating effective morpholino and mRNA molecules with controls for injection, and optimizing injection conditions.

Characterization of nAChRs in Nematostella vectensis supports neuronal and non-neuronal roles in the cnidarian-bilaterian common ancestor.

BACKGROUND: Nicotinic and muscarinic acetylcholine receptors likely evolved in the cnidarian-bilaterian common ancestor. Both receptor families are best known for their role at chemical synapses in bilaterian animals, but they also have described roles as non-neuronal signaling receptors within the bilaterians. It is not clear when either of the functions for nicotinic or muscarinic receptors evolved. Previous studies in cnidarians suggest that acetylcholine's neuronal role existed prior to the cnidarian-bilaterian divergence, but did not address potential non-neuronal functions. To determine the origins of neuronal and non-neuronal functions of nicotinic acetylcholine receptors, we investigated the phylogenetic position of cnidarian acetylcholine receptors, characterized the spatiotemporal expression patterns of nicotinic receptors in N. vectensis, and compared pharmacological studies in N. vectensis to the previous work in other cnidarians. RESULTS: Consistent with described activity in other cnidarians, treatment with acetylcholine-induced tentacular contractions in the cnidarian sea anemone N. vectensis. Phylogenetic analysis suggests that the N. vectensis genome encodes 26 nicotinic (nAChRs) and no muscarinic (mAChRs) acetylcholine receptors and that nAChRs independently radiated in cnidarian and bilaterian linages. The namesake nAChR agonist, nicotine, induced tentacular contractions similar to those observed with acetylcholine, and the nAChR antagonist mecamylamine suppressed tentacular contractions induced by both acetylcholine and nicotine. This indicated that tentacle contractions are in fact mediated by nAChRs. Nicotine also induced the contraction of radial muscles, which contract as part of the peristaltic waves that propagate along the oral-aboral axis of the trunk. Radial contractions and peristaltic waves were suppressed by mecamylamine. The ability of nicotine to mimic acetylcholine responses, and of mecamylamine to suppress acetylcholine and nicotine-induced contractions, supports a neuronal function for acetylcholine in cnidarians. Examination of the spatiotemporal expression of N. vectensis nAChRs (NvnAChRs) during development and in juvenile polyps identified that NvnAChRs are expressed in neurons, muscles, gonads, and large domains known to be consistent with a role in developmental patterning. These patterns are consistent with nAChRs functioning in both a neuronal and non-neuronal capacity in N. vectensis. CONCLUSION: Our data suggest that nAChR receptors functioned at chemical synapses in N. vectensis to regulate tentacle contraction. Similar responses to acetylcholine are well documented in cnidarians, suggesting that the neuronal function represents an ancestral role for nAChRs. Expression patterns of nAChRs are consistent with both neuronal and non-neuronal roles for acetylcholine in cnidarians. Together, these observations suggest that both neuronal and non-neuronal functions for the ancestral nAChRs were present in the cnidarian-bilaterian common ancestor. Thus, both roles described in bilaterian species likely arose at or near the base of nAChR evolution.

Cnidarian Zic Genes.

To understand the ancestral and evolved roles of zic homologs, it is important to reconstruct the putative roles of ancient zic homologs in the animal phylogeny. Most studies of zic genes have been conducted in model systems that are members of the bilaterian phylum. However, two additional phyla have zic homologs encoded in their genomes. The three animal phyla that contain zic homologs all share a common ancestor and collectively are termed the parahoxozoans (cnidarians (corals, sea anemones, and jellyfish), placozoans (Trichoplax adhaerens), and bilaterians (chordates, insects, nematodes, annelids, echinoderms, etc.). In this chapter we briefly discuss our understanding of zic genes in the parahoxozoans with a particular focus on how expression of cnidarian zic homologs in the medusozoan Hydra vulgaris and the anthozoan Nematostella vectensis informs our understanding of the putative ancestral roles zic homologs played in the cnidarian-bilaterian common ancestor.

Characterization of NvLWamide-like neurons reveals stereotypy in Nematostella nerve net development.

The organization of cnidarian nerve nets is traditionally described as diffuse with randomly arranged neurites that show minimal reproducibility between animals. However, most observations of nerve nets are conducted using cross-reactive antibodies that broadly label neurons, which potentially masks stereotyped patterns produced by individual neuronal subtypes. Additionally, many cnidarians species have overt structures such as a nerve ring, suggesting higher levels of organization and stereotypy exist, but mechanisms that generated that stereotypy are unknown. We previously demonstrated that NvLWamide-like is expressed in a small subset of the Nematostella nerve net and speculated that observing a few neurons within the developing nerve net would provide a better indication of potential stereotypy. Here we document NvLWamide-like expression more systematically. NvLWamide-like is initially expressed in the typical neurogenic salt and pepper pattern within the ectoderm at the gastrula stage, and expression expands to include endodermal salt and pepper expression at the planula larval stage. Expression persists in both ectoderm and endoderm in adults. We characterized our NvLWamide-like::mCherry transgenic reporter line to visualize neural architecture and found that NvLWamide-like is expressed in six neural subtypes identifiable by neural morphology and location. Upon completing development the numbers of neurons in each neural subtype are minimally variable between animals and the projection patterns of each subtype are consistent. Furthermore, between the juvenile polyp and adult stages the number of neurons for each subtype increases. We conclude that development of the Nematostella nerve net is stereotyped between individuals. Our data also imply that one aspect of generating adult cnidarian nervous systems is to modify the basic structural architecture generated in the juvenile by increasing neural number proportionally with size.

A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning.

Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

The cellular and molecular basis of cnidarian neurogenesis.

Neurogenesis initiates during early development and it continues through later developmental stages and in adult animals to enable expansion, remodeling, and homeostasis of the nervous system. The generation of nerve cells has been analyzed in detail in few bilaterian model organisms, leaving open many questions about the evolution of this process. As the sister group to bilaterians, cnidarians occupy an informative phylogenetic position to address the early evolution of cellular and molecular aspects of neurogenesis and to understand common principles of neural development. Here we review studies in several cnidarian model systems that have revealed significant similarities and interesting differences compared to neurogenesis in bilaterian species, and between different cnidarian taxa. Cnidarian neurogenesis is currently best understood in the sea anemone Nematostella vectensis, where it includes epithelial neural progenitor cells that express transcription factors of the soxB and atonal families. Notch signaling regulates the number of these neural progenitor cells, achaete-scute and dmrt genes are required for their further development and Wnt and BMP signaling appear to be involved in the patterning of the nervous system. In contrast to many vertebrates and Drosophila, cnidarians have a high capacity to generate neurons throughout their lifetime and during regeneration. Utilizing this feature of cnidarian biology will likely allow gaining new insights into the similarities and differences of embryonic and regenerative neurogenesis. The use of different cnidarian model systems and their expanding experimental toolkits will thus continue to provide a better understanding of evolutionary and developmental aspects of nervous system formation. WIREs Dev Biol 2017, 6:e257. doi: 10.1002/wdev.257 For further resources related to this article, please visit the WIREs website.

MAPK signaling is necessary for neurogenesis in Nematostella vectensis.

BACKGROUND: The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood. RESULTS: We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages. CONCLUSIONS: Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.

The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration.

Reverse genetics and next-generation sequencing unlocked a new era in biology. It is now possible to identify an animal(s) with the unique biology most relevant to a particular question and rapidly generate tools to functionally dissect that biology. This review highlights the rise of one such novel model system, the starlet sea anemone Nematostella vectensis. Nematostella is a cnidarian (corals, jellyfish, hydras, sea anemones, etc.) animal that was originally targeted by EvoDevo researchers looking to identify a cnidarian animal to which the development of bilaterians (insects, worms, echinoderms, vertebrates, mollusks, etc.) could be compared. Studies in Nematostella have accomplished this goal and informed our understanding of the evolution of key bilaterian features. However, Nematostella is now going beyond its intended utility with potential as a model to better understand other areas such as regenerative biology, EcoDevo, or stress response. This review intends to highlight key EvoDevo insights from Nematostella that guide our understanding about the evolution of axial patterning mechanisms, mesoderm, and nervous systems in bilaterians, as well as to discuss briefly the potential of Nematostella as a model to better understand the relationship between development and regeneration. Lastly, the sum of research to date in Nematostella has generated a variety of tools that aided the rise of Nematostella to a viable model system. We provide a catalogue of current resources and techniques available to facilitate investigators interested in incorporating Nematostella into their research. WIREs Dev Biol 2016, 5:408-428. doi: 10.1002/wdev.222 For further resources related to this article, please visit the WIREs website.

Functional Characterization of Cnidarian HCN Channels Points to an Early Evolution of Ih.

HCN channels play a unique role in bilaterian physiology as the only hyperpolarization-gated cation channels. Their voltage-gating is regulated by cyclic nucleotides and phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of HCN channels provides the depolarizing current in response to hyperpolarization that is critical for intrinsic rhythmicity in neurons and the sinoatrial node. Additionally, HCN channels regulate dendritic excitability in a wide variety of neurons. Little is known about the early functional evolution of HCN channels, but the presence of HCN sequences in basal metazoan phyla and choanoflagellates, a protozoan sister group to the metazoans, indicate that the gene family predates metazoan emergence. We functionally characterized two HCN channel orthologs from Nematostella vectensis (Cnidaria, Anthozoa) to determine which properties of HCN channels were established prior to the emergence of bilaterians. We find Nematostella HCN channels share all the major functional features of bilaterian HCNs, including reversed voltage-dependence, activation by cAMP and PIP2, and block by extracellular Cs+. Thus bilaterian-like HCN channels were already present in the common parahoxozoan ancestor of bilaterians and cnidarians, at a time when the functional diversity of voltage-gated K+ channels was rapidly expanding. NvHCN1 and NvHCN2 are expressed broadly in planulae and in both the endoderm and ectoderm of juvenile polyps.

Ether-à-go-go family voltage-gated K+ channels evolved in an ancestral metazoan and functionally diversified in a cnidarian-bilaterian ancestor.

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K(+) channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg(2+) in Eag channels and Ca(2+) in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype.

Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr.

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K(+) channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K(+) channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.

Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation.

BACKGROUND: Cellular differentiation is a critical process during development of multicellular animals that must be tightly controlled in order to avoid precocious differentiation or failed generation of differentiated cell types. Research in flies, vertebrates, and nematodes has led to the identification of a conserved role for Notch signaling as a mechanism to regulate cellular differentiation regardless of tissue/cell type. Notch signaling can occur through a canonical pathway that results in the activation of hes gene expression by a complex consisting of the Notch intracellular domain, SuH, and the Mastermind co-activator. Alternatively, Notch signaling can occur via a non-canonical mechanism that does not require SuH or activation of hes gene expression. Regardless of which mechanism is being used, high Notch activity generally inhibits further differentiation, while low Notch activity promotes differentiation. Flies, vertebrates, and nematodes are all bilaterians, and it is therefore unclear if Notch regulation of differentiation is a bilaterian innovation, or if it represents a more ancient mechanism in animals. RESULTS: To reconstruct the ancestral function of Notch signaling we investigate Notch function in a non-bilaterian animal, the sea anemone Nematostella vectensis (Cnidaria). Morpholino or pharmacological knockdown of Nvnotch causes increased expression of the neural differentiation gene NvashA. Conversely, overactivation of Notch activity resulting from overexpression of the Nvnotch intracellular domain or by overexpression of the Notch ligand Nvdelta suppresses NvashA. We also knocked down or overactivated components of the canonical Notch signaling pathway. We disrupted NvsuH with morpholino or by overexpressing a dominant negative NvsuH construct. We saw no change in expression levels for Nvhes genes or NvashA. Overexpression of Nvhes genes did not alter NvashA expression levels. Lastly, we tested additional markers associated with neuronal differentiation and observed that non-canonical Notch signaling broadly suppresses neural differentiation in Nematostella. CONCLUSIONS: We conclude that one ancestral role for Notch in metazoans was to regulate neural differentiation. Remarkably, we found no evidence for a functional canonical Notch pathway during Nematostella embryogenesis, suggesting that the non-canonical hes-independent Notch signaling mechanism may represent an ancestral Notch signaling pathway.