Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin, Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. While the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2, piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression past metamorphosis. F0 animals were raised to sexual maturity and spawned to determine germline integration, transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrated transgene transmission through the germline, the first example of a germline transgenic sea urchin, and indeed of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility, and efficiency of sea urchin research.

Solute carrier (SLC) expression reveals skeletogenic cell diversity.

Within the developing embryo is a microcosm of cell type diversity. Single cell RNA-sequencing (scRNA-seq) is used to reveal cell types, typically by grouping cells according to their gene regulatory states. However, both across and within these regulatory states are additional layers of cellular diversity represented by the differential expression of genes that govern cell function. Here, we analyzed scRNA-seq data representing the late gastrula stage of Strongylocentrotus purpuratus (purple sea urchin) to understand the patterning of transporters belonging to the ABC and SLC families. These transporters handle diverse substrates from amino acids to signaling molecules, nutrients and xenobiotics. Using transporter-based clustering, we identified unique transporter patterns that are both shared across cell lineages, as well as those that were unique to known cell types. We further explored three patterns of transporter expression in mesodermal cells including secondary mesenchyme cells (pigment cells and blastocoelar cells) and skeletogenic cells (primary mesenchyme cells). The results revealed the enrichment of SMTs potentially involved in nutrient absorption (SLC5A9, SLC7A11, SLC35F3, and SLC52A3) and skeletogenesis (SLC9A3, SLC13A2/3/5, and SLC39A13) in pigment cells and blastocoelar cells respectively. The results indicated that the strategy of clustering by cellular activity can be useful for discovering cellular populations that would otherwise remain obscured.

Wnt/ß-catenin signaling induces axial elasticity patterns of Hydra extracellular matrix.

The extracellular matrix (ECM) plays crucial roles in animal development and diseases. Here, we report that Wnt/ß-catenin signaling induces the ECM remodeling during Hydra axis formation. We determined the micro- and nanoscopic arrangement of fibrillar type I collagen along Hydra's body axis using high-resolution microscopy and X-ray scattering. Elasticity mapping of the ECM ex vivo revealed distinctive elasticity patterns along the body axis. A proteomic analysis of the ECM showed that these elasticity patterns correlate with a gradient-like distribution of metalloproteases along the body axis. Activation of the Wnt/ß-catenin pathway in wild-type and transgenic animals alters these patterns toward low ECM elasticity patterns. This suggests a mechanism whereby high protease activity under control of Wnt/ß-catenin signaling causes remodeling and softening of the ECM. This Wnt-dependent spatiotemporal coordination of biochemical and biomechanical cues in ECM formation was likely a central evolutionary innovation for animal tissue morphogenesis.

Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.

The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxide­sensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.