Highlights d mob4 of the STRIPAK complex limits body size in planarians d wnt1+ posterior signaling-center cells promote tail size during regeneration d mob4 limits differentiation of wnt1+ pole cells from stem cells d Production of specialized signaling cells regulates organ size in regeneration

The complexity of cell types and states revealed by single-cell RNAseq atlases presents a challenge for the systematic analysis of fate determinants using traditional screening methodologies. Differentiation in the planarian Schmidtea mediterranea exemplifies this problem, as these animals continuously produce over 100 differentiated cell types for homeostasis and regeneration using neoblast adult pluripotent stem cells. The signaling factors enabling neoblast self-renewal and selective differentiation of these many fates are still incompletely understood. We developed a method using high-throughput expression profiling by qPCR and whole-animal RNAseq to simultaneously assess numerous cell fate markers as the phenotypic readout in large-scale RNAi screens. Applying this method, we performed an RNAi screen of 400 kinases, receptors, and other regulatory molecules to reveal specific functions for 30 previously unknown factors in neoblast biology. 17 genes were required for neoblast maintenance, including factors likely involved in cell-cycle regulation, nutrient sensing, and chromatin modification. Multidimensional expression information additionally revealed several specific regulators of other neoblast activities, including a mink1 kinase regulating global neoblast differentiation, the energy responsive kinase adenylate kinase-2 regulating intestine specification within the neoblast population, an RNA acetyl transferase nat10 regulating epidermal differentiation, and a pak1 kinase restricting neoblast localization to prevent tissue outgrowths. These results identify several new regulators of neoblast activities and demonstrate the applicability of expression-based screening for systematic analysis of stem cell phenotypes in whole animals.

Integration of positional information across body axes is likely critical for whole-body regeneration to define the territories of missing tissue in three dimensions with fidelity. The body-wall musculature in planarians expresses patterning factors regulating the anteroposterior, dorsoventral, and mediolateral axes, but how this information coordinates is not fully understood. Using analysis of scRNAseq data, we identify pitx and islet transcription factors as regulators of a dorsal muscle midline signaling center expressing a the BMP/Activin decoy receptor bambi-2. Dorsal midline cells are comprised of longitudinal muscle and are required for blastema formation in fragments lacking a pre-existing midline. In laterally amputated animals regenerating an entirely new midline, bambi-2+ cells are initially formed at the dorsal-most region of the excised tissue fragment, then dynamically spread and ultimately reset to restore bilateral symmetry, rather than forming at the initial geometric mediolateral midpoint. We further identify a system of dorsoventral Netrin and Netrin receptor signals expressed from body-wall muscle that control midline identity and blastema morphology. Ventral and laterally expressed netrins -1, -4, and -5 signal via dorsally-enriched netrin repulsion receptors unc-5C, unc-5E, and dcc-2, which together limit mediolateral spread of bambi-2+ dorsal midline muscle and enable muscle targeting across the midline. Our results suggest a model in which ventral determinants dictate mediolateral information important for blastema patterning.

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