The ability to regenerate missing body parts exists throughout the animal kingdom. Positional information is crucial for regeneration, but how it is harboured and used by differentiated tissues is poorly understood. In planarians, positional information has been identified from study of phenotypes caused by RNA interference in which the wrong tissues are regenerated. For example, inhibition of the Wnt signalling pathway leads to regeneration of heads in place of tails1,2,3. Characterization of these phenotypes has led to the identification of position control genes (PCGs)—genes that are expressed in a constitutive and regional manner and are associated with patterning. Most PCGs are expressed within planarian muscle4; however, how muscle is specified and how different muscle subsets affect regeneration is unknown. Here we show that different muscle fibres have distinct regulatory roles during regeneration in the planarian Schmidtea mediterranea. myoD is required for formation of a specific muscle cell subset: the longitudinal fibres, oriented along the anterior–posterior axis. Loss of longitudinal fibres led to complete regeneration failure because of defects in regeneration initiation. A different transcription factor-encoding gene, nkx1-1, is required for the formation of circular fibres, oriented along the medial–lateral axis. Loss of circular fibres led to a bifurcated anterior–posterior axis with fused heads forming in single anterior blastemas. Whereas muscle is often viewed as a strictly contractile tissue, these findings reveal that different muscle types have distinct and specific regulatory roles in wound signalling and patterning to enable regeneration.
Gene Expression Omnibus
NCBI Reference Sequence
We thank N. Watson and A. Mahowald for transmission electron microscopy; S. LoCascio for eye resections; C.-C. Chen for V5277; and M. Fedorovsky for illustrations. We acknowledge support from NIH R01GM080639 and the Eleanor Schwartz Charitable Foundation. P.W.R. is an Investigator of the HHMI and an associate member of the Broad Institute of Harvard and MIT.
Extended data figures
Immunostaining using 6G10 and V5277 antibodies show different layers of muscle fibres in the planarian BWM. The video shows the layers of muscle fibres on the ventral side of the animal, and goes from the outer layer (closer to the epidermis) to the inner layer (closer to the parenchyma). First in the video, the circular muscle fibres can be observed, next the diagonal fibres and last the longitudinal fibres are observed. Muscle fibres from a 5-7 mm animal have been previously estimated to measure between 150-200 μm of length (Baguñà, J. & Romero, R. Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84, 181-194, (1981)).
Immunostaining using 6G10 and V5277 antibodies shows loss of the longitudinal fiber layer in a myoD(RNAi) animal. Similar to SI video 1, the confocal z-stack was taken on the ventral side of the animal. From the epidermis into the animal parenchyma the circular and diagonal muscle fibre layers can be observed but very few longitudinal muscle fibres are left intact after inhibition of myoD.
Immunostaining using 6G10 and V5277 antibodies shows loss of the circular fibre layer (first layer observed in the video) in a nkx1-1(RNAi) animal. Similar to SI video 1, the confocal z-stack was taken on the ventral side of the animal. From the epidermis into the animal parenchyma few circular muscle fibres are left intact in the outer layer, but diagonal and longitudinal muscle layers are maintained after inhibition of nkx1-1.
FISH shows glutamate decarboxylase (gd) RNA probe in green, DAPI in gray. The confocal z-stack was taken from the ventral side of the animal. In green, the arc of gd+ cells can be observed.
FISH shows ectopic glutamate decarboxylase expression in green, DAPI in gray. A wider brain with a small forming brain lobe can be observed.
Immunostaining using 6G10 and V5277 antibodies shows loss of both circular fibre (first layer shown in the video) and longitudinal fibre (last layer shown in the video) layers and disorganization of BWM in a double myoD, nkx1-1(RNAi) animal.