Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis.

With a surprisingly complex genome and an ever-expanding genetic toolkit, the sea anemone Nematostella vectensis has become a powerful model system for the study of both development and whole-body regeneration. Here we provide the most current protocols for short-hairpin RNA (shRNA )-mediated gene knockdown and CRISPR/Cas9-targeted mutagenesis in this system. We further show that a simple Klenow reaction followed by in vitro transcription allows for the production of gene-specific shRNAs and single guide RNAs (sgRNAs) in a fast, affordable, and readily scalable manner. Together, shRNA knockdown and CRISPR/Cas9-targeted mutagenesis allow for rapid screens of gene function as well as the production of stable mutant lines that enable functional genetic analysis throughout the Nematostella life cycle.

Hedgehog signaling is required for endomesodermal patterning and germ cell development in the sea anemone Nematostella vectensis.

Two distinct mechanisms for primordial germ cell (PGC) specification are observed within Bilatera: early determination by maternal factors or late induction by zygotic cues. Here we investigate the molecular basis for PGC specification in Nematostella, a representative pre-bilaterian animal where PGCs arise as paired endomesodermal cell clusters during early development. We first present evidence that the putative PGCs delaminate from the endomesoderm upon feeding, migrate into the gonad primordia, and mature into germ cells. We then show that the PGC clusters arise at the interface between hedgehog1 and patched domains in the developing mesenteries and use gene knockdown, knockout and inhibitor experiments to demonstrate that Hh signaling is required for both PGC specification and general endomesodermal patterning. These results provide evidence that the Nematostella germline is specified by inductive signals rather than maternal factors, and support the existence of zygotically-induced PGCs in the eumetazoan common ancestor.

Feeding-dependent tentacle development in the sea anemone Nematostella vectensis.

In cnidarians, axial patterning is not restricted to embryogenesis but continues throughout a prolonged life history filled with unpredictable environmental changes. How this developmental capacity copes with fluctuations of food availability and whether it recapitulates embryonic mechanisms remain poorly understood. Here we utilize the tentacles of the sea anemone Nematostella vectensis as an experimental paradigm for developmental patterning across distinct life history stages. By analyzing over 1000 growing polyps, we find that tentacle progression is stereotyped and occurs in a feeding-dependent manner. Using a combination of genetic, cellular and molecular approaches, we demonstrate that the crosstalk between Target of Rapamycin (TOR) and Fibroblast growth factor receptor b (Fgfrb) signaling in ring muscles defines tentacle primordia in fed polyps. Interestingly, Fgfrb-dependent polarized growth is observed in polyp but not embryonic tentacle primordia. These findings show an unexpected plasticity of tentacle development, and link post-embryonic body patterning with food availability.

Cell-Cycle-Coupled Oscillations in Apical Polarity and Intercellular Contact Maintain Order in Embryonic Epithelia.

Throughout animals, embryonic cells must ultimately organize into polarized epithelial layers that provide the structural basis for gastrulation or subsequent developmental events [1]. Precisely how this primary epithelium maintains continuous integrity during rapid and repeated cell divisions has never been directly addressed, particularly in cases where early cleavages are driven in synchrony. Representing the early-branching non-bilaterian phylum Cnidaria, embryos of the sea anemone Nematostella vectensis undergo rapid synchronous cell divisions and ultimately give rise to a diploblastic epithelial body plan after gastrulation [2, 3]. Here, using live imaging of apical polarity proteins in Nematostella embryos, we demonstrate that cell polarity is established by the four-cell stage and then reiteratively lost during subsequent mitoses, correlating with transient adhesion disengagement and dramatic deformations of embryonic morphology. Intriguingly, the re-establishment of polarity and adhesion during each interphase is associated with a process of whole-embryo compaction analogous to that observed in mammals [4-7]. Because similar protein dynamics are observed in dividing epithelial cells in Drosophila melanogaster, we propose that cell-cycle-coupled oscillations in apical polarity may be conserved throughout Metazoa.

Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function.

Treacher Collins syndrome (TCS) is a congenital disorder of craniofacial development arising from mutations in TCOF1, which encodes the nucleolar phosphoprotein Treacle. Haploinsufficiency of Tcof1 perturbs mature ribosome biogenesis, resulting in stabilization of p53 and the cyclin G1-mediated cell-cycle arrest that underpins the specificity of neuroepithelial apoptosis and neural crest cell hypoplasia characteristic of TCS. Here we show that inhibition of p53 prevents cyclin G1-driven apoptotic elimination of neural crest cells while rescuing the craniofacial abnormalities associated with mutations in Tcof1 and extending life span. These improvements, however, occur independently of the effects on ribosome biogenesis; thus suggesting that it is p53-dependent neuroepithelial apoptosis that is the primary mechanism underlying the pathogenesis of TCS. Our work further implies that neuroepithelial and neural crest cells are particularly sensitive to cellular stress during embryogenesis and that suppression of p53 function provides an attractive avenue for possible clinical prevention of TCS craniofacial birth defects and possibly those of other neurocristopathies.