Updated single cell reference atlas for the starlet anemone Nematostella vectensis.

BACKGROUND: The recent combination of genomics and single cell transcriptomics has allowed to assess a variety of non-conventional model organisms in much more depth. Single cell transcriptomes can uncover hidden cellular complexity and cell lineage relationships within organisms. The recent developmental cell atlases of the sea anemone Nematostella vectensis, a representative of the basally branching Cnidaria, has provided new insights into the development of all cell types (Steger et al Cell Rep 40(12):111370, 2022; Sebé-Pedrós et al. Cell 173(6):1520-1534.e20). However, the mapping of the single cell reads still suffers from relatively poor gene annotations and a draft genome consisting of many scaffolds. RESULTS: Here we present a new wildtype resource of the developmental single cell atlas, by re-mapping of sequence data first published in Steger et al. (2022) and Cole et al. (Nat Commun 14(1):1747, 2023), to the new chromosome-level genome assembly and corresponding gene models in Zimmermann et al. (Nat Commun 14, 8270 (2023). https://doi.org/10.1038/s41467-023-44080-7 ). We expand the pre-existing dataset through the incorporation of additional sequence data derived from the capture and sequencing of cell suspensions from four additional samples: 24 h gastrula, 2d planula, an inter-parietal region of the bodywall from a young unsexed animal, and another adult mesentery from a mature male animal. CONCLUSION: Our analyses of the full cell-state inventory provide transcriptomic signatures for 127 distinct cell states, of which 47 correspond to neuroglandular subtypes. We also identify two distinct putatively immune-related transcriptomic profiles that segregate between the inner and outer cell layers. Furthermore, the new gene annotation Nv2 has markedly improved the mapping on the single cell transcriptome data and will therefore be of great value for the community and anyone using the dataset.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

Successful adaption of a forensic toxicological screening workflow employing nontargeted liquid chromatography-tandem mass spectrometry to water analysis.

Forensic toxicology and environmental water analysis share the common interest and responsibility in ensuring comprehensive and reliable confirmation of drugs and pharmaceutical compounds in samples analyzed. Dealing with similar analytes, detection and identification techniques should be exchangeable between scientific disciplines. Herein, we demonstrate the successful adaption of a forensic toxicological screening workflow employing nontargeted LC/MS/MS under data-dependent acquisition control and subsequent database search to water analysis. The main modification involved processing of an increased sample volume with SPE (500 mL vs. 1-10 mL) to reach LODs in the low ng/L range. Tandem mass spectra acquired with a qTOF instrument were submitted to database search. The targeted data mining strategy was found to be sensitive and specific; automated search produced hardly any false results. To demonstrate the applicability of the adapted workflow to complex samples, 14 wastewater effluent samples collected on seven consecutive days at the local wastewater-treatment plant were analyzed. Of the 88,970 fragment ion mass spectra produced, 8.8% of spectra were successfully assigned to one of the 1040 reference compounds included in the database, and this enabled the identification of 51 compounds representing important illegal drugs, members of various pharmaceutical compound classes, and metabolites thereof.

Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain.

The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter-motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that tmt-opsins co-express with val-opsins, known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports "sensory-inter-motorneurons" as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra, possibly providing the endogenous equivalent to an optogenetic machinery.

Pbx3 and Meis1 cooperate through multiple mechanisms to support Hox-induced murine leukemia.

Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia.

Muscle cell-type diversification is driven by bHLH transcription factor expansion and extensive effector gene duplications.

Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the inner cell layer into the neural ectoderm contributes to the evolution of a novel muscle cell type. Thus, we conclude that extensive transcription factor gene duplications and co-option of effector modules act as an evolutionary mechanism underlying cell type diversification during metazoan evolution.

DAXX drives de novo lipogenesis and contributes to tumorigenesis.

Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX's SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX's C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip.

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

Single-cell transcriptomics identifies conserved regulators of neuroglandular lineages.

Communication in bilaterian nervous systems is mediated by electrical and secreted signals; however, the evolutionary origin and relation of neurons to other secretory cell types has not been elucidated. Here, we use developmental single-cell RNA sequencing in the cnidarian Nematostella vectensis, representing an early evolutionary lineage with a simple nervous system. Validated by transgenics, we demonstrate that neurons, stinging cells, and gland cells arise from a common multipotent progenitor population. We identify the conserved transcription factor gene SoxC as a key upstream regulator of all neuroglandular lineages and demonstrate that SoxC knockdown eliminates both neuronal and secretory cell types. While in vertebrates and many other bilaterians neurogenesis is largely restricted to early developmental stages, we show that in the sea anemone, differentiation of neuroglandular cells is maintained throughout all life stages, and follows the same molecular trajectories from embryo to adulthood, ensuring lifelong homeostasis of neuroglandular cell lineages.

Pattern regulation in a regenerating jellyfish.

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around 'hubs' which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber 'spokes'. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/ß-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.

The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterized by distinct transcriptome signatures reflecting abrupt life-cycle transitions and all deploy a mixture of phylogenetically old and new genes. Medusa-specific transcription factors, including many with bilaterian orthologues, associate with diverse neurosensory structures. Compared to Clytia, the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content evolution and sequence evolution. Absence of expression and gene loss among Clytia orthologues of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys the ancestral cnidarian-bilaterian transcription factor gene complement.

HoxA9 transforms murine myeloid cells by a feedback loop driving expression of key oncogenes and cell cycle control genes.

Ectopic expression of the oncogenic transcription factor HoxA9 is a major cause of acute myeloid leukemia (AML). Here, we demonstrate that HoxA9 is a specific substrate of granule proteases. Protease knockout allowed the comprehensive determination of genome-wide HoxA9 binding sites by chromatin immunoprecipitation sequencing in primary murine cells and a human AML cell line. The kinetics of enhancer activity and transcription rates in response to alterations of an inducible HoxA9 were determined. This permitted identification of HoxA9-controlled enhancers and promoters, allocation to their respective transcription units, and discrimination against HoxA9-bound, but unresponsive, elements. HoxA9 triggered an elaborate positive-feedback loop that drove expression of the complete Hox-A locus. In addition, it controlled key oncogenic transcription factors Myc and Myb and directly induced the cell cycle regulators Cdk6 and CyclinD1, as well as telomerase, drawing the essential blueprint for perturbation of proliferation by leukemogenic HoxA9 expression.