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.

A cis-regulatory change underlying the motor neuron-specific loss of Ebf expression in immotile tunicate larvae.

Many species in the tunicate family Molgulidae have independently lost their swimming larval form and instead develop as tailless, immotile larvae. These larvae do not develop structures that are essential for swimming such as the notochord, otolith, and tail muscles. However, little is known about neural development in these nonswimming larvae. Here, we studied the patterning of the Motor Ganglion (MG) of Molgula occulta, a nonswimming species. We found that spatial patterns of MG neuron regulators in this species are conserved, compared with species with swimming larvae, suggesting that the gene networks regulating their expression are intact despite the loss of swimming. However, expression of the key motor neuron regulatory gene Ebf (Collier/Olf/EBF) was reduced in the developing MG of M. occulta when compared with molgulid species with swimming larvae. This was corroborated by measuring allele-specific expression of Ebf in hybrid embryos from crosses of M. occulta with the swimming species M. oculata. Heterologous reporter construct assays in the model tunicate species Ciona robusta revealed a specific cis-regulatory sequence change that reduces expression of Ebf in the MG, but not in other cells. Taken together, these data suggest that MG neurons are still specified in M. occulta larvae, but their differentiation might be impaired due to reduction of Ebf expression levels.

The ctenophore genome and the evolutionary origins of neural systems.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets.

ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.

Phylogenomics offers resolution of major tunicate relationships.

Tunicata, a diverse clade of approximately 3000 described species of marine, filter-feeding chordates, is of great interest to researchers because tunicates are the closest living relatives of vertebrates and they facilitate comparative studies of our own biology. The group also includes numerous invasive species that cause considerable economic damage and some species of tunicates are edible. Despite their diversity and importance, relationships among major lineages of Tunicata are not completely resolved. Here, we supplemented public data with transcriptomes from seven species spanning the diversity of Tunicata and conducted phylogenomic analyses on data sets of up to 798 genes. Sensitivity analyses were employed to examine the influences of reducing compositional heterogeneity and branch-length heterogeneity. All analyses maximally supported a monophyletic Tunicata within Olfactores (Vertebrata + Tunicata). Within Tunicata, all analyses recovered Appendicularia sister to the rest of Tunicata and confirmed (with maximal support) that Thaliacea is nested within Ascidiacea. Stolidobranchia is the sister taxon to all other tunicates except Appendicularia. In most analyses, phlebobranch tunicates were recovered paraphyletic with respect to Aplousobranchia. Support for this topology varied but was strong in some cases. However, when only the 50 best genes based on compositional heterogeneity were analysed, we recovered Phlebobranchia and Aplousobranchia reciprocally monophyletic with strong support, consistent with most traditional morphology-based hypotheses. Examination of internode certainty also cast doubt on results of phlebobranch paraphyly, which may be due to limited taxon sampling. Taken together, these results provide a higher-level phylogenetic framework for our closest living invertebrate relatives.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Head regeneration in hemichordates is not a strict recapitulation of development.

BACKGROUND: Head or anterior body part regeneration is commonly associated with protostome, but not deuterostome invertebrates. However, it has been shown that the solitary hemichordate Ptychodera flava possesses the remarkable capacity to regenerate their entire nervous system, including their dorsal neural tube and their anterior head-like structure, or proboscis. Hemichordates, also known as acorn worms, are marine invertebrate deuterostomes that have retained chordate traits that were likely present in the deuterostome ancestor, placing these animals in a vital position to study regeneration and chordate evolution. All acorn worms have a tripartite body plan, with an anterior proboscis, middle collar region, and a posterior trunk. The collar houses a hollow, dorsal neural tube in ptychoderid hemichordates and numerous chordate genes involved in brain and spinal cord development are expressed in a similar anterior-posterior spatial arrangement along the body axis. RESULTS: We have examined anterior regeneration in the hemichordate Ptychodera flava and report the spatial and temporal morphological changes that occur. Additionally, we have sequenced, assembled, and analyzed the transcriptome for eight stages of regenerating P. flava, revealing significant differential gene expression between regenerating and control animals. CONCLUSIONS: Importantly, we have uncovered developmental steps that are regeneration-specific and do not strictly follow the embryonic program. Developmental Dynamics 245:1159-1175, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Metamorphosis in solitary ascidians.

Embryonic and postembryonic development in ascidians have been studied for over a century, but it is only in the last 10 years that the complex molecular network involved in coordinating postlarval development and metamorphosis has started to emerge. In most ascidians, the transition from the larval to the sessile juvenile/adult stage, or metamorphosis, requires a combination of environmental and endogenous signals and is characterized by coordinated global morphogenetic changes that are initiated by the adhesion of the larvae. Cloney was the first to describe cellular events of ascidians' metamorphosis in 1978 and only recently elements of the molecular regulation of this crucial developmental step have been revealed. This review aims to present a thorough view of this crucial developmental step by combining recent molecular data to the already established cellular events.

Guidelines for the nomenclature of genetic elements in tunicate genomes.

Tunicates are invertebrate members of the chordate phylum, and are considered to be the sister group of vertebrates. Tunicates are composed of ascidians, thaliaceans, and appendicularians. With the advent of inexpensive high-throughput sequencing, the number of sequenced tunicate genomes is expected to rise sharply within the coming years. To facilitate comparative genomics within the tunicates, and between tunicates and vertebrates, standardized rules for the nomenclature of tunicate genetic elements need to be established. Here we propose a set of nomenclature rules, consensual within the community, for predicted genes, pseudogenes, transcripts, operons, transcriptional cis-regulatory regions, transposable elements, and transgenic constructs. In addition, the document proposes guidelines for naming transgenic and mutant lines.

Evolution and development of budding by stem cells: ascidian coloniality as a case study.

The evolution of budding in metazoans is not well understood on a mechanistic level, but is an important developmental process. We examine the evolution of coloniality in ascidians, contrasting the life histories of solitary and colonial forms with a focus on the cellular and developmental basis of the evolution of budding. Tunicates are an excellent group to study colonial transitions, as all solitary larvae develop with determinant and invariant cleavage patterns, but colonial species show robust developmental flexibility during larval development. We propose that acquiring new stem cell lineages in the larvae may be a preadaptation necessary for the evolution of budding. Brooding in colonial ascidians allows increased egg size, which in turn allows greater flexibility in the specification of cells and cell numbers in late embryonic and pre-metamorphic larval stages. We review hypotheses for changes in stem cell lineages in colonial species, describe what the current data suggest about the evolution of budding, and discuss where we believe further studies will be most fruitful.

MicroRNAs support the monophyly of enteropneust hemichordates.

Understanding the evolutionary history of deuterostomes requires elucidating the phylogenetic interrelationships amongst the constituent taxa. Although the monophyly and interrelationships among the three principal groups-the chordates, the echinoderms, and the hemichordates-are well established, as are the internal relationships among the echinoderm and chordate taxa, the interrelationships among the principal groups of hemichordates-the harrimaniid enteropneusts, the ptychoderid enteropneusts, and the pterobranchs-remain unresolved. Depending on the study some find enteropneusts paraphyletic with pterobranchs (e.g., Cephalodiscus) more closely related to the harrimaniid enteropneusts (e.g., Saccoglossus) than either are to the ptychoderid enteropneusts (e.g., Ptychodera), whereas other studies support a monophyletic Enteropneusta. To try and resolve between these two competing hypotheses, we turned to microRNAs, small ∼22 nt non-coding RNA genes that have been shown to shed insight into particularly difficult phylogenetic questions. Using deep sequencing we characterized the small RNA repertoires of two hemichordate species, Cephalodiscus hodgsoni and Ptychodera flava, and the crinoid echinoderm Antedon mediterranea, and combined our results with the described complements of the hemichordate Saccoglossus kowalevskii, the sea urchin Strongylocentrotus purpuratus, and the starfish Patiria miniata. Our data unambiguously support the monophyly of Enteropneusts as S. kowalevskii shares 12 miRNA sequences with P. flava that are not present in the C. hodgsoni or A. mediterranea libraries, and have never been reported from another metazoan taxon. Thus, these data resolve the phylogenetic position of pterobranchs, ultimately allowing for a better understanding of body plan evolution throughout the deuterostomes.

Developmental mode influences diversification in ascidians.

Ascidian species (Tunicata: Ascidiacea) usually have tailed, hatching tadpole larvae. In several lineages, species have evolved larvae that completely lack any tail tissues and are unable to disperse actively. Some tailless species hatch, but some do not hatch before going through metamorphosis. We show here that ascidian species with the highest speciation rates are those with the largest range sizes and tailed hatching larval development. We use methods for examining diversification in binary characters across a posterior distribution of trees, and show that mode of larval development predicts geographical range sizes. Conversely, we find that species with the least dispersive larval development (tailless, non-hatching) have the lowest speciation rates and smallest geographical ranges. Our speciation rate results are contrary to findings from sea urchins and snails examined in the fossil record, and further work is necessary to reconcile these disparate results.

Loss of collagen gene expression in the notochord of the tailless tunicate Molgula occulta.

In tunicates, several species in the Molgulidae family have convergently lost the tailed, swimming larval body plan, including the morphogenesis of the notochord, a major chordate-defining trait. Through the comparison of tailless M. occulta and a close relative, the tailed species M. oculata, we show that notochord-specific expression of the Collagen Type I/II Alpha (Col1/2a) gene appears to have been lost specifically in the tailless species. Using CRISPR/Cas9-mediated mutagenesis in the tailed laboratory model tunicate Ciona robusta, we demonstrate that Col1/2a plays a crucial role in the convergent extension of notochord cells during tail elongation. Our results suggest that the expression of Col1/2a in the notochord, although necessary for its morphogenesis in tailed species, is dispensable for tailless species. This loss is likely a result of the accumulation of cis-regulatory mutations in the absence of purifying selective pressure. More importantly, the gene itself is not lost, likely due to its roles in other developmental processes, including during the adult stage. Our study further confirms the Molgulidae as an interesting family in which to study the evolutionary loss of tissue-specific expression of indispensable genes.

Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.

Early lineage specification of long-lived germline precursors in the colonial ascidian Botryllus schlosseri.

In many taxa, germline precursors segregate from somatic lineages during embryonic development and are irreversibly committed to gametogenesis. However, in animals that can propagate asexually, germline precursors can originate in adults. Botryllus schlosseri is a colonial ascidian that grows by asexual reproduction, and on a weekly basis regenerates all somatic and germline tissues. Embryonic development in solitary ascidians is the classic example of determinative specification, and we are interested in both the origins and the persistence of stem cells responsible for asexual development in colonial ascidians. In this study, we characterized vasa as a putative marker of germline precursors. We found that maternally deposited vasa mRNA segregates early in development to a posterior lineage of cells, suggesting that germline formation is determinative in colonial ascidians. In adults, vasa expression was observed in the gonads, as well as in a population of mobile cells scattered throughout the open circulatory system, consistent with previous transplantation/reconstitution results. vasa expression was dynamic during asexual development in both fertile and infertile adults, and was also enriched in a population of stem cells. Germline precursors in juveniles could contribute to gamete formation immediately upon transplantation into fertile adults, thus vasa expression is correlated with the potential for gamete formation, which suggests that it is a marker for embryonically specified, long-lived germline progenitors. Transient vasa knockdown did not have obvious effects on germline or somatic development in adult colonies, although it did result in a profound heterochrony, suggesting that vasa might play a homeostatic role in asexual development.

Phylogeography and reproductive variation of the poecilogonous polychaete Boccardia proboscidea (Annelida: Spionidae) along the West Coast of North America.

The ability to produce more than one kind of offspring, or poecilogony, is a striking example of reproductive variability. Traditionally, larval nutrition has been classified as a dichotomy: if offspring obtain nutrition from their mothers (lecithotrophy), there is lower fecundity and greater chance of offspring survival than when they get their nutrition from plankton (planktotrophy). The polychaete Boccardia proboscidea (Spionidae) produces both types of embryos using three different reproductive strategies. In this study, we examined the roles of genetic history and phenotypic plasticity on explaining natural variation in B. proboscidea along the Pacific coast of the United States using two genetic mitochondrial markers, 16S rDNA and Cyt b, and common garden experiments. These data show a single North American West Coast network that is structured, geographically, by the well-documented biogeographic break near Point Conception, California. The southern group within this network covers a smaller range, but has larger haplotype diversity, than the northern group. Some individuals differing in reproductive type had the same haplotype, indicating independence of these features; however, differences between laboratory and field data suggest additional geographic variation within one of the reproductive types. Females from higher latitudes provide offspring with larger supplies of extra embryonic nutrition than females from southern latitudes. Results herein suggest that both genetic history and developmental plasticity are playing a role in the maintenance of this reproductive polymorphism.

Transitional chordates and vertebrate origins: Tunicates.

The Origin of Chordates has fascinated scientists from the time of Charles Darwin's publication "Descent of Man" in 1871. For over 100 years, it was accepted that chordates evolved from tunicates, our sessile invertebrate sister group. However, genomic and embryonic analyses have shown that lancelets have a body plan and genome much more like vertebrates than do tunicates. In 2000, we proposed a worm-like hypothesis of chordate origins, and genomic and embryonic studies in the past 20 years have supported this hypothesis. This hypothesis contends that the deuterostome ancestor was worm-like, with gill slits, very much like a chordate. In contrast, tunicates have a very derived adult body plan that evolved independently. Here, we review the current understanding of deuterostome phylogeny and supporting evidence for the relationships within each phylum. Then we discuss our hypothesis for chordate origins and evidence to support it. We explore some of the evolutionary changes that ascidians have made to their adult body plan and some of the key gene regulatory networks that have been elucidated in Ciona. Finally, we end with insights that we have gained from studying tailless ascidians for the past 30 years. We've found that differentiation genes, at the end of the gene regulatory networks, become pseudogenes and nonfunctional, even though they are still expressed in tailless ascidians. We expect that eventually these pseudogenes will not be expressed and the ascidian larval body plan is abandoned, leaving the embryo to develop directly into an adult.

The Degenerate Tale of Ascidian Tails.

Ascidians are invertebrate chordates, with swimming chordate tadpole larvae that have distinct heads and tails. The head contains the small brain, sensory organs, including the ocellus (light) and otolith (gravity) and the presumptive endoderm, while the tail has a notochord surrounded by muscle cells and a dorsal nerve cord. One of the chordate features is a post-anal tail. Ascidian tadpoles are nonfeeding, and their tails are critical for larval locomotion. After hatching the larvae swim up toward light and are carried by the tide and ocean currents. When competent to settle, ascidian tadpole larvae swim down, away from light, to settle and metamorphose into a sessile adult. Tunicates are classified as chordates because of their chordate tadpole larvae; in contrast, the sessile adult has a U-shaped gut and very derived body plan, looking nothing like a chordate. There is one group of ascidians, the Molgulidae, where many species are known to have tailless larvae. The Swalla Lab has been studying the evolution of tailless ascidian larvae in this clade for over 30 years and has shown that tailless larvae have evolved independently several times in this clade. Comparison of the genomes of two closely related species, the tailed Molgula oculata and tailless Molgula occulta reveals much synteny, but there have been multiple insertions and deletions that have disrupted larval genes in the tailless species. Genomics and transcriptomics have previously shown that there are pseudogenes expressed in the tailless embryos, suggesting that the partial rescue of tailed features in their hybrid larvae is due to the expression of intact genes from the tailed parent. Yet surprisingly, we find that the notochord gene regulatory network is mostly intact in the tailless M. occulta, although the notochord does not converge and extend and remains as an aggregate of cells we call the "notoball." We expect that eventually many of the larval gene networks will become evolutionarily lost in tailless ascidians and the larval body plan abandoned, with eggs developing directly into an adult. Here we review the current evolutionary and developmental evidence on how the molgulids lost their tails.

Whole body regeneration in a colonial ascidian, Botrylloides violaceus.

Colonial ascidians are the only chordates to undergo whole body regeneration (WBR), the ability to form an entirely new individual from the peripheral vasculature. Here we describe WBR in Botrylloides violaceus, a colonial ascidian that reliably regenerates after ablation of all zooids and buds of young colonies. During early regeneration several buds develop within the tunic vasculature, but only one continues development into a complete zooid. We describe some of the first events of vascular budding leading to the vesicle stage with phase contrast microscopy, time-lapse video recording and detailed histological studies of regenerating colonies. The first conspicuous stage of vascular budding is when a single-layered sphere of cells becomes enclosed by vascular epithelium. We report the appearance of Piwi-positive cells in hemocytes surrounding the regenerates. We observed an increase of proliferating cell nuclear antigen (PCNA)-positive cells in circulatory hemocytes in late regenerates, and found double-labeled nuclear expression with Piwi in a subset of large circulatory cells. We rarely found Piwi or PCNA in differentiating tissues during vascular budding, suggesting that cells that form the epithelial tissues during budding and WBR originate mostly from circulatory hemocyte precursors. We propose that multiple stem cell types are circulating within B. violaceus and that they undergo proliferation in the peripheral vasculature before differentiating into epithelial tissues for all three germ layers during WBR.