Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution.

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

Molecular characterization of nervous system organization in the hemichordate acorn worm Saccoglossus kowalevskii.

Hemichordates are an important group for investigating the evolution of bilaterian nervous systems. As the closest chordate outgroup with a bilaterally symmetric adult body plan, hemichordates are particularly informative for exploring the origins of chordates. Despite the importance of hemichordate neuroanatomy for testing hypotheses on deuterostome and chordate evolution, adult hemichordate nervous systems have not been comprehensively described using molecular techniques, and classic histological descriptions disagree on basic aspects of nervous system organization. A molecular description of hemichordate nervous system organization is important for both anatomical comparisons across phyla and for attempts to understand how conserved gene regulatory programs for ectodermal patterning relate to morphological evolution in deep time. Here, we describe the basic organization of the adult hemichordate Saccoglossus kowalevskii nervous system using immunofluorescence, in situ hybridization, and transgenic reporters to visualize neurons, neuropil, and key neuronal cell types. Consistent with previous descriptions, we found the S. kowalevskii nervous system consists of a pervasive nerve plexus concentrated in the anterior, along with nerve cords on both the dorsal and ventral side. Neuronal cell types exhibited clear anteroposterior and dorsoventral regionalization in multiple areas of the body. We observed spatially demarcated expression patterns for many genes involved in synthesis or transport of neurotransmitters and neuropeptides but did not observe clear distinctions between putatively centralized and decentralized portions of the nervous system. The plexus shows regionalized structure and is consistent with the proboscis base as a major site for information processing rather than the dorsal nerve cord. In the trunk, there is a clear division of cell types between the dorsal and ventral cords, suggesting differences in function. The absence of neural processes crossing the basement membrane into muscle and extensive axonal varicosities suggest that volume transmission may play an important role in neural function. These data now facilitate more informed neural comparisons between hemichordates and other groups, contributing to broader debates on the origins and evolution of bilaterian nervous systems.

Integrating Complex Life Cycles in Comparative Developmental Biology.

The goal of comparative developmental biology is identifying mechanistic differences in embryonic development between different taxa and how these evolutionary changes have led to morphological and organizational differences in adult body plans. Much of this work has focused on direct-developing species in which the adult forms straight from the embryo and embryonic modifications have direct effects on the adult. However, most animal lineages are defined by indirect development, in which the embryo gives rise to a larval body plan and the adult forms by transformation of the larva. Historically, much of our understanding of complex life cycles is viewed through the lenses of ecology and zoology. In this review, we discuss the importance of establishing developmental rather than morphological or ecological criteria for defining developmental mode and explicitly considering the evolutionary implications of incorporating complex life cycles into broad developmental comparisons of embryos across metazoans.

Induced Immune Reaction in the Acorn Worm, Saccoglossus kowalevskii, Informs the Evolution of Antiviral Immunity.

Evolutionary perspectives on the deployment of immune factors following infection have been shaped by studies on a limited number of biomedical model systems with a heavy emphasis on vertebrate species. Although their contributions to contemporary immunology cannot be understated, a broader phylogenetic perspective is needed to understand the evolution of immune systems across Metazoa. In our study, we leverage differential gene expression analyses to identify genes implicated in the antiviral immune response of the acorn worm hemichordate, Saccoglossus kowalevskii, and place them in the context of immunity evolution within deuterostomes-the animal clade composed of chordates, hemichordates, and echinoderms. Following acute exposure to the synthetic viral double-stranded RNA analog, poly(I:C), we show that S. kowalevskii responds by regulating the transcription of genes associated with canonical innate immunity signaling pathways (e.g., nuclear factor κB and interferon regulatory factor signaling) and metabolic processes (e.g., lipid metabolism), as well as many genes without clear evidence of orthology with those of model species. Aggregated across all experimental time point contrasts, we identify 423 genes that are differentially expressed in response to poly(I:C). We also identify 147 genes with altered temporal patterns of expression in response to immune challenge. By characterizing the molecular toolkit involved in hemichordate antiviral immunity, our findings provide vital evolutionary context for understanding the origins of immune systems within Deuterostomia.

Alternative Chemistries for Free Radical-Initiated Targeting and Immobilization.

Stimuli-responsive biomaterials are an emerging strategy that leverage common pathophysiological triggers to target drug delivery to limit or avoid toxic side effects. Native free radicals, such as reactive oxygen species (ROS), are widely upregulated in many pathological states. We have previously demonstrated that native ROS are capable of crosslinking and immobilizing acrylated polyethylene glycol diacrylate (PEGDA) networks and coupled payloads in tissue mimics, providing evidence for a potential targeting mechanism. To build on these promising results, we evaluated PEG dialkenes and dithiols as alternative polymer chemistries for targeting. The reactivity, toxicity, crosslinking kinetics, and immobilization potential of PEG dialkenes and dithiols were characterized. Both the alkene and thiol chemistries crosslinked in the presence of ROS, generating high molecular weight polymer networks that immobilized fluorescent payloads in tissue mimics. Thiols were especially reactive and even reacted with acrylates in the absence of free radicals, and this motivated us to explore a two-phase targeting approach. Delivering thiolated payloads in a second phase, after the initial polymer net formation, allowed greater control over the payload dosing and timing. Two-phase delivery combined with a library of radical-sensitive chemistries can enhance the versatility and flexibility of this free radical-initiated platform delivery system.

Testing specificity and sensitivity of wastewater-based epidemiology for detecting SARS-CoV-2 in four communities on Vancouver Island, Canada.

We report wastewater surveillance of the spread of SARS-CoV-2 based upon 24-h composite influent samples taken weekly from four wastewater treatment plants (WWTP) on Vancouver Island, BC, Canada between January 3, 2021 and July 10, 2021. Samples were analyzed by reverse transcription quantitative polymerase chain reaction targeting the N1 and N2 gene fragments of SARS-CoV-2 and a region of the replication associate protein of the pepper mottle mosaic virus (PMMoV) serving as endemic control. Only a small proportion of samples had quantifiable levels of N1 or N2. Overall case rates are weakly correlated with the concentration (gene copies/L) and with the flux of viral material influent to the WWTP (gene copies/day); the latter accounts for influent flow variations. Poisson multimodal rank correlation accounts for differences between the four WWTP and shows a significant correlation with a significant positive intercept. Receiver operator characteristics (ROC) analysis confirms a cut-off of cases based on amplified/not-amplified experimental data. At the optimal cut point of 19 (N1) or 17 (N2) cases/week/100,000 the sensitivity and specificity is about 75% for N1 and 67% for N2.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum.

BACKGROUND: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79-88, 2012) to answer these questions. RESULTS: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. CONCLUSIONS: Cell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

Saccoglossus kowalevskii: Evo-devo insights from the mud.

Hemichordates have long been recognized as a critical group for addressing hypotheses of chordate origins. Historically this was due to anatomical traits that resembled those of chordates, most strikingly the dorsolateral gill slits. As molecular data and phylogenetic analyses were found to support a close phylogenetic relationship between hemichordates and chordates within the deuterostomes, interest was revived in hemichordates. In particular, Saccoglossus kowalevskii has been developed as a molecular model to represent hemichordate developmental biology. Herein, we highlight the considerations when choosing a particular species to study and the challenges we encountered when developing S. kowalevskii. We discuss our findings and how method and tool development enabled them, and how we envision expanding our repertoire of molecular tools in the future. Establishing a new model organism comes with many obstacles-from identifying a reliable season to collect animals, to developing modern molecular techniques. The Saccoglossus research community has benefited greatly from the collaborations and teamwork established over the years. As a result, Saccoglossus is well positioned to contribute to a new century of evolutionary developmental (evo-devo) research.

Molecular evidence for a single origin of ultrafiltration-based excretory organs.

Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1-3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia.

Molecular insights into deuterostome evolution from hemichordate developmental biology.

Hemichordates, along with echinoderms and chordates, belong to the lineage of bilaterians called the deuterostomes. Their phylogenetic position as an outgroup to chordates provides an opportunity to investigate the evolutionary origins of the chordate body plan and reconstruct ancestral deuterostome characters. The body plans of the hemichordates and chordates are organizationally divergent making anatomical comparisons very challenging. The developmental underpinnings of animal body plans are often more conservative than the body plans they regulate, and offer a novel data set for making comparisons between morphologically divergent body architectures. Here I review the hemichordate developmental data generated over the past 20 years that further test hypotheses of proposed morphological affinities between the two taxa, but also compare the conserved anteroposterior, dorsoventral axial patterning programs and germ layer specification programs. These data provide an opportunity to determine which developmental programs are ancestral deuterostome or bilaterian innovations, and which ones occurred in stem chordates or vertebrates representing developmental novelties of the chordate body plan.

Enrichment of circulating tumor-derived extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 µL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

Neural anatomy of echinoid early juveniles and comparison of nervous system organization in echinoderms.

The echinoderms are a phylum of marine deuterostomes characterized by the pentaradial (five fold) symmetry of their adult bodies. Due to this unusual body plan, adult echinoderms have long been excluded from comparative analyses aimed at understanding the origin and evolution of deuterostome nervous systems. Here, we investigated the neural anatomy of early juveniles of representatives of three of the five echinoderm classes: the echinoid Paracentrotus lividus, the asteroid Patiria miniata, and the holothuroid Parastichopus parvimensis. Using whole mount immunohistochemistry and confocal microscopy, we found that the nervous system of echinoid early juveniles is composed of three main structures: a basiepidermal nerve plexus, five radial nerve cords connected by a circumoral nerve ring, and peripheral nerves innervating the appendages. Our whole mount preparations further allowed us to obtain thorough descriptions of these structures and of several innervation patterns, in particular at the level of the appendages. Detailed comparisons of the echinoid juvenile nervous system with those of asteroid and holothuroid juveniles moreover supported a general conservation of the main neural structures in all three species, including at the level of the appendages. Our results support the previously proposed hypotheses for the existence of two neural units in echinoderms: one consisting of the basiepidermal nerve plexus to process sensory stimuli locally and one composed of the radial nerve cords and the peripheral nerves constituting a centralized control system. This study provides the basis for more in-depth comparisons of the echinoderm adult nervous system with those of other animals, in particular hemichordates and chordates, to address the long-standing controversies about deuterostome nervous system evolution.

Untangling posterior growth and segmentation by analyzing mechanisms of axis elongation in hemichordates.

The trunk is a key feature of the bilaterian body plan. Despite spectacular morphological diversity in bilaterian trunk anatomies, most insights into trunk development are from segmented taxa, namely arthropods and chordates. Mechanisms of posterior axis elongation (PAE) and segmentation are tightly coupled in arthropods and vertebrates, making it challenging to differentiate between the underlying developmental mechanisms specific to each process. Investigating trunk elongation in unsegmented animals facilitates examination of mechanisms specific to PAE and provides a different perspective for testing hypotheses of bilaterian trunk evolution. Here we investigate the developmental roles of canonical Wnt and Notch signaling in the hemichordate Saccoglossus kowalevskii and reveal that both pathways play key roles in PAE immediately following the completion of gastrulation. Furthermore, our functional analysis of the role of Brachyury is supportive of a Wnt-Brachyury feedback loop during PAE in S. kowalevskii, establishing this key regulatory interaction as an ancestral feature of deuterostomes. Together, our results provide valuable data for testing hypotheses of bilaterian trunk evolution.

Free radical-mediated targeting and immobilization of coupled payloads.

Targeted drug delivery is a promising approach to enhance the accumulation of therapies in diseased tissues while limiting off-site effects. Ligand-receptor interactions are traditionally identified to deliver therapies, and although specific, this can be costly and often suffers from limited sensitivity. An emerging approach is to target intermediary species that modulate disease progression. Here, we propose novel methods of targeting therapies by using native free radicals as a homing signal. Elevated concentrations of free radicals are a characteristic comorbidity of many different diseases. In polymer chemistry, free radicals are frequently used to initiate crosslinking reactions. We proposed that free radicals elevated in injury sites are capable of inducing crosslinking of acrylate groups on polymer chains. Coupling payloads to the polymer then allow for specific targeting of therapies to areas with elevated free radicals. We demonstrate in vitro proof-of-principle of this approach. Reactive oxygen species (ROS) initiated crosslinking of acrylated PEGs, which immobilized a fluorescent payload within tissue mimics. The cross-linking efficiency and immobilization potential varied with the polymer chain length, suggesting that a tuneable platform can be achieved. Together these results provide promising proof-of-concept for using free radicals to specifically target and sustain nearly endless payloads to disease sites.

The cadherin-catenin complex is necessary for cell adhesion and embryogenesis in Nematostella vectensis.

The cadherin-catenin complex is a conserved, calcium-dependent cell-cell adhesion module that is necessary for normal development and the maintenance of tissue integrity in bilaterian animals. Despite longstanding evidence of a deep ancestry of calcium-dependent cell adhesion in animals, the requirement of the cadherin-catenin complex to coordinate cell-cell adhesion has not been tested directly in a non-bilaterian organism. Here, we provide the first analysis of classical cadherins and catenins in the Starlet Sea Anemone, Nematostella vectensis. Gene expression, protein localization, siRNA-mediated knockdown of α-catenin, and calcium-dependent cell aggregation assays provide evidence that a bonafide cadherin-catenin complex is present in the early embryo, and that α-catenin is required for normal embryonic development and the formation of cell-cell adhesions between cells dissociated from whole embryos. Together these results support the hypothesis that the cadherin-catenin complex was likely a complete and functional cell-cell adhesion module in the last common cnidarian-bilaterian ancestor. SUMMARY STATEMENT: Embryonic manipulations and ex vivo adhesion assays in the sea anemone, Nematostella vectensis, indicate that the necessity of the cadherin-catenin complex for mediating cell-cell adhesion is deeply conserved in animal evolution.

I-SceI Meganuclease-mediated transgenesis in the acorn worm, Saccoglossus kowalevskii.

Hemichordates are a phylum of marine invertebrate deuterostomes that are closely related to chordates, and represent one of the most promising models to provide insights into early deuterostome evolution. The genome of the hemichordate, Saccoglossus kowalevskii, reveals an extensive set of non-coding elements conserved across all three deuterostome phyla. Functional characterization and cross-phyla comparisons of these putative regulatory elements will enable a better understanding of enhancer evolution, and subsequently how changes in gene regulation give rise to morphological innovation. Here, we describe an efficient method of transgenesis for the characterization of non-coding elements in S. kowalevskii. We first test the capacity of an I-SceI transgenesis system to drive ubiquitous or regionalized gene expression, and to label specific cell types. Finally, we identified a minimal promoter that can be used to test the capacity of putative enhancers in S. kowalevskii. This work demonstrates that this I-SceI transgenesis technique, when coupled with an understanding of chromatin accessibility, can be a powerful tool for studying how evolutionary changes in gene regulatory mechanisms contributed to the diversification of body plans in deuterostomes.

The development and metamorphosis of the indirect developing acorn worm Schizocardium californicum (Enteropneusta: Spengelidae).

BACKGROUND: Enteropneusts are benthic marine invertebrates that belong to the deuterostome phylum Hemichordata. The two main clades of enteropneusts are defined by differences in early life history strategies. In the Spengelidae and Ptychoderidae, development is indirect via a planktotrophic tornaria larva. In contrast, development in the Harrimanidae is direct without an intervening larval life history stage. Most molecular studies in the development and evolution of the enteropneust adult body plan have been carried out in the harrimanid Saccoglossus kowalevskii. In order to compare these two developmental strategies, we have selected the spengelid enteropneust Schizocardium californicum as a suitable indirect developing species for molecular developmental studies. Here we describe the methods for adult collecting, spawning and larval rearing in Schizocardium californicum, and describe embryogenesis, larval development, and metamorphosis, using light microscopy, immunocytochemistry and confocal microscopy. RESULTS: Adult reproductive individuals can be collected intertidally and almost year-round. Spawning can be triggered by heat shock and large numbers of larvae can be reared through metamorphosis under laboratory conditions. Gastrulation begins at 17 h post-fertilization (hpf) and embryos hatch at 26 hpf as ciliated gastrulae. At 3 days post-fertilization (dpf), the tornaria has a circumoral ciliary band, mouth, tripartite digestive tract, protocoel, larval muscles and a simple serotonergic nervous system. The telotroch develops at 5 dpf. In the course of 60 days, the serotonergic nervous system becomes more elaborate, the posterior coeloms develop, and the length of the circumoral ciliary band increases. At the end of the larval stage, larval muscles disappear, gill slits form, and adult muscles develop. Metamorphosis occurs spontaneously when the larva reaches its maximal size (ca. 3 mm), and involves loss and reorganization of larval structures (muscles, nervous system, digestive tract), as well as development of adult structures (adult muscles, tripartite body organization). CONCLUSIONS: This study will enable future research in S. californicum to address long standing questions related to the evolution of axial patterning mechanisms, germ layer induction, neurogenesis and neural patterning, the mechanisms of metamorphosis, the relationships between larval and adult body plans, and the evolution of metazoan larval forms.

Risks of hypoxia and acidification in the high energy coastal environment near Victoria, Canada's untreated municipal sewage outfalls.

Wastewater disposal often has deleterious impacts on the receiving environment. Low dissolved oxygen levels are particularly concerning. Here, we investigate the impacts on dissolved oxygen and carbon chemistry of screened municipal wastewater in the marine waters off Victoria, Canada. We analyzed data from undersea moorings, ship-based monitoring, and remotely-operated vehicle video. We used these observations to construct a two-layer model of the nearfield receiving environment. Despite the lack of advanced treatment, dissolved oxygen levels near the outfalls were well above a 62 µmol kg-1 hypoxic threshold. Furthermore, the impact on water column oxygen at the outfall is likely <2 µmol kg-1. Dissolved inorganic carbon is not elevated and pH not depressed compared to the surrounding region. Strong tidal currents and cold, well-ventilated waters give Victoria's marine environment a high assimilative capacity for organic waste. However, declining oxygen levels offshore put water near the outfall at risk of future hypoxia.

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development.

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.

A thermoreversible, photocrosslinkable collagen bio-ink for free-form fabrication of scaffolds for regenerative medicine.

As a biomaterial, collagen has been used throughout tissue engineering and regenerative medicine. Collagen is native to the body, is highly biocompatible, and naturally promotes cell adhesion and regeneration. However, collagen fibers and the inherent weak mechanical properties of collagen hydrogels interfere with further development of collagen as a bio-ink. Herein, we demonstrate the use of a modified type-I collagen, collagen methacrylamide (CMA), as a fibril-forming bio-ink for free-form fabrication of scaffolds. Like collagen, CMA can self-assemble into a fibrillar hydrogel at physiological conditions. In contrast, CMA is photocrosslinkable and thermoreversible, and photocrosslinking eliminates thermoreversibility. Free-form fabrication of CMA was performed through self-assembly of the CMA hydrogel, photocrosslinking the structure of interest using a photomask, and cooling the entire hydrogel, which results in cold-melting of unphotocrosslinked regions. Printed hydrogels had a resolution on the order of ~350 µm, and can be fabricated with or without cells and maintain viability or be further processed into freeze-dried sponges, all while retaining pattern fidelity. A subcutaneous implant study confirmed the biocompatibility of CMA in comparison to collagen. Free-form fabrication of CMA allows for printing of macroscale, customized scaffolds with good pattern fidelity and can be implemented with relative ease for continued research and development of collagen-based scaffolds in tissue engineering.