Mechanisms determining final organ size are poorly understood. Animals undergoing regeneration or ongoing adult growth are likely to require sustained and robust mechanisms to achieve and maintain appropriate sizes. Planarians, well known for their ability to undergo whole-body regeneration using pluripotent adult stem cells of the neoblast population, can reversibly scale body size over an order of magnitude by controlling cell number. Using quantitative analysis, we showed that after injury planarians perfectly restored brain:body proportion by increasing brain cell number through epimorphosis or decreasing brain cell number through tissue remodeling (morphallaxis), as appropriate. We identified a pathway controlling a brain size set-point that involves feedback inhibition between wnt11-6/wntA/wnt4a and notum, encoding conserved antagonistic signaling factors expressed at opposite brain poles. wnt11-6/wntA/wnt4a undergoes feedback inhibition through canonical Wnt signaling but is likely to regulate brain size in a non-canonical pathway independently of beta-catenin-1 and APC. Wnt/Notum signaling tunes numbers of differentiated brain cells in regenerative growth and tissue remodeling by influencing the abundance of brain progenitors descended from pluripotent stem cells, as opposed to regulating cell death. These results suggest that the attainment of final organ size might be accomplished by achieving a balance of positional signaling inputs that regulate the rates of tissue production.

Cnidaria (sea anemones, jellyfish, corals and hydra) form a close sister group to Bilateria. Within this clade, the sea anemone Nematostella vectensis has emerged as a slow evolving model for investigating characteristics of the cnidarian-bilaterian common ancestor, which diverged near the Cambrian explosion. Here, using long read sequencing and high throughput chromosome conformation capture, we generate high quality chromosome-level genome assemblies for N. vectensis and the closely related edwardsiid sea anemone, Scolanthus callimorphus. In both cases we find a robust set of 15 chromosomes comprising a stable linkage group detectable within all major clades of sequenced cnidarian genomes. Further, both genomes show remarkable chromosomal conservation with chordates. In contrast with Bilateria, we report that extended Hox and NK gene clusters are chromosomally linked but do not retain a tight spatial conservation. Accordingly, there is a lack of evidence for topologically associated domains, which have been implicated in the evolutionary pressure to retain tight microsyntenic gene clusters. We also uncover ultra-conserved noncoding elements at levels previously undetected in non-chordate lineages. Both genomes are accessible through an actively updated genome browser and database at https://simrbase.stowers.org

Most animals undergo homeostatic tissue maintenance, yet those capable of robust regeneration in adulthood use mechanisms significantly overlapping with homeostasis. Here we show in planarians that modulations to body-wide patterning systems shift the target site for eye regeneration while still enabling homeostasis of eyes outside this region. The uncoupling of homeostasis and regeneration, which can occur during normal positional rescaling after axis truncation, is not due to altered injury signaling or stem cell activity, nor specific to eye tissue. Rather, pre-existing tissues, which are misaligned with patterning factor expression domains, compete with properly located organs for incorporation of migratory progenitors. These observations suggest that patterning factors determine sites of organ regeneration but do not solely determine the location of tissue homeostasis. These properties provide candidate explanations for how regeneration integrates pre-existing tissues and how regenerative abilities could be lost in evolution or development without eliminating long-term tissue maintenance and repair.

Draft genome sequences of non-bilaterian species have provided important insights into the evolution of the metazoan gene repertoire. However, there is little information about the evolution of gene clusters, genome architectures and karyotypes during animal evolution. Here we report chromosome-level genome assemblies of two related anthozoan cnidarians, the sea anemones, Nematostella vectensis and Scolanthus callimorphus. We find a robust set of 15 chromosomes with a clear one-to-one correspondence of the chromosomes between the two species. We show that, in contrast to Bilateria, Hox and NK clusters of investigated cnidarians are disintegrated, indicating that microsynteny conservation is largely lost. In line with that, we find no evidence for topologically associated domains, suggesting fundamental difference in long-range gene regulation compared to vertebrates. However, both sea anemone genomes show remarkable chromosomal conservation with other cnidarians, several bilaterians and the sponge Ephydatia muelleri, allowing us to reconstruct the putative cnidarian and metazoan chromosomes, consisting of 19 and 16 ancestral linkage groups, respectively. These data suggest that large parts of the ancestral metazoan genome have been retained in chromosomes of some extant lineages, yet, higher order gene regulation may have evolved only after the cnidarian-bilaterian split.

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, shRNAknockdown 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.