Adult regeneration restores patterning of orthogonal body axes after damage in a post-embryonic context. Planarians regenerate using distinct body-wide signals primarily regulating each axis dimension: anteroposterior Wnts, dorsoventral BMP, and mediolateral Wnt5 and Slit determinants. How regeneration can coordinate perpendicular tissue axes without symmetry-breaking embryonic events is not fully understood. Here, we report that the planarian dorsoventral regulator bmp4 suppresses the posterior determinant wnt1 to provide patterning input to the anteroposterior axis. Double-FISH identified distinct anteroposterior domains within dorsal midline muscle that express either bmp4 or wnt1. Homeostatic inhibition bmp4 and smad1 expanded the wnt1 expression anteriorly, while elevation of BMP signaling through nog1;nog2 RNAi reduced the wnt1 expression domain and elevated bmp4 expression. Homeostatic BMP signal perturbation broadly affected anteroposterior identity as measured by expression of posterior Wnt pathway factors, and caused mislocalization of AP-regionalized pharynx progenitors, without strongly affecting expression domains of anterior regulators. Additionally, wnt1 inhibition elevated bmp4 expression in the tip of the tail. Therefore, dorsal BMP signals and posterior wnt1 mutually antagonize for patterning the tail. Furthermore, homeostatic bmp4 RNAi caused medial expansion of the lateral determinant wnt5 and reduced expression of the medial regulator slit. By contrast, nog1;nog2 RNAi restricted wnt5 expression. Double RNAi of bmp4 and wnt5 resulted in lateral ectopic eye phenotypes, suggesting bmp4 acts upstream of wnt5 to pattern the mediolateral axis. These results indicate bmp4 controls dorsoventral information and also, through suppression of Wnt signals, influences anteroposterior and mediolateral identity. Based on related functions across vertebrates and Cnidarians, Wnt and BMP cross-regulation could form an ancient mechanism for coordinating orthogonal axis patterning.

Adult regeneration restores patterning of orthogonal body axes after damage in a post-embryonic context. Planarians regenerate using distinct body-wide signals primarily regulating each axis dimension: anteroposterior Wnts, dorsoventral BMP, and mediolateral Wnt5 and Slit determinants. How regeneration can consistently form perpendicular tissue axes without symmetry-breaking embryonic events is unknown, and could either occur using fully independent, or alternatively, integrated signals defining each dimension. Here, we report that the planarian dorsoventral regulator bmp4 suppresses the posterior determinant wnt1 to pattern the anteroposterior axis. Double-FISH identified distinct anteroposterior domains within dorsal midline muscle that express either bmp4 or wnt1 . Homeostatic inhibition bmp4 and smad1 expanded the wnt1 expression anteriorly, while elevation of BMP signaling through nog1;nog2 RNAi reduced the wnt1 expression domain. BMP signal perturbation broadly affected anteroposterior identity as measured by expression of posterior Wnt pathway factors, without affecting head regionalization. Therefore, dorsal BMP signals broadly limit posterior identity. Furthermore, bmp4 RNAi caused medial expansion of the lateral determinant wnt5 and reduced expression of the medial regulator slit . Double RNAi of bmp4 and wnt5 resulted in lateral ectopic eye phenotypes, suggesting bmp4 acts upstream of wnt5 to pattern the mediolateral axis. Therefore, bmp4 acts at the top of a patterning hierarchy both to control dorsoventral information and also, through suppression of Wnt signals, to regulate anteroposterior and mediolateral identity. These results reveal that adult pattern formation involves integration of signals controlling individual orthogonal axes.

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.

Wnt signaling regulates primary body axis formation across the Metazoa, with high Wnt signaling specifying posterior identity. Whether a common Wnt-driven transcriptional program accomplishes this broad role is poorly understood. We identified genes acutely affected after Wnt signaling inhibition in the posterior of two regenerative species, the planarian Schmidtea mediterranea and the acoel Hofstenia miamia, which are separated by >550 million years of evolution. Wnt signaling was found to maintain positional information in muscle and regional gene expression in multiple differentiated cell types. sp5, Hox genes, and Wnt pathway components are down-regulated rapidly after β-catenin RNAi in both species. Brachyury, a vertebrate Wnt target, also displays Wnt-dependent expression in Hofstenia. sp5 inhibits trunk gene expression in the tail of planarians and acoels, promoting separate tail-trunk body domains. A planarian posterior Hox gene, Post-2d, promotes normal tail regeneration. We propose that common regulation of a small gene set-Hox, sp5, and Brachyury-might underlie the widespread utilization of Wnt signaling in primary axis patterning across the Bilateria.

Negative regulators of adult neurogenesis are of particular interest as targets to enhance neuronal repair, but few have yet been identified. Planarians can regenerate their entire CNS using pluripotent adult stem cells, and this process is robustly regulated to ensure that new neurons are produced in proper abundance. Using a high-throughput pipeline to quantify brain chemosensory neurons, we identify the conserved tyrosine kinase tec-1 as a negative regulator of planarian neuronal regeneration. tec-1RNAi increased the abundance of several CNS and PNS neuron subtypes regenerated or maintained through homeostasis, without affecting body patterning or non-neural cells. Experiments using TUNEL, BrdU, progenitor labeling, and stem cell elimination during regeneration indicate tec-1 limits the survival of newly differentiated neurons. In vertebrates, the Tec kinase family has been studied extensively for roles in immune function, and our results identify a novel role for tec-1 as negative regulator of planarian adult neurogenesis.