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First published online 17 October 2007
doi: 10.1242/dev.007138

Development 134, 4043-4051 (2007)
Published by The Company of Biologists 2007
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BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

Peter W. Reddien1, Adam L. Bermange2,*, Adrienne M. Kicza1 and Alejandro Sánchez Alvarado2,{dagger}

1 MIT Biology, Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02138, USA.
2 Howard Hughes Medical Institute, Department of Neurobiology and Anatomy, University of Utah School of Medicine, 401 MREB, 20N 1900E, Salt Lake City, UT 84132, USA.


Figure 1
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  		Fig. 1. smedolloid-1 is expressed in small dorsally located cells and smedbmp4-1 is expressed in cells along the dorsal midline. (A) Schematic of a planarian depicting body regions and terminology used throughout this manuscript. Anterior, up. Dotted line indicates the dorsal midline. Solid lines indicate surgical amputations. (B,C) Dorsal (B) and Ventral (C) views of wild-type animals labeled with the smedolloid-1 riboprobe. An enlarged view of a head region is shown on the right in B. (D) A smedolloid-1(RNAi) animal showed no labeling in an in situ hybridization with a smedolloid-1 riboprobe. Scale bar: 0.1 mm. (E) Wild-type animals were labeled with the smedbmp4-1 riboprobe. px, pharynx. Arrowhead, smedbmp4-1 signal at the anterior end of the pharynx. Scale bar: 0.5 mm. (F) Section of a wild-type animal showing expression of smedbmp4-1 in sub-epidermal, dorsally localized cells. Following whole-mount in situ hybridization with a smedbmp4-1 riboprobe, animals were sectioned as previously described (Reddien et al., 2005bGo). In both panels, dorsal is to the right. Left panel, DAPI staining; right panel, Nomarski optics. E, epidermis. Scale bar: 10 µm. (G) A smedbmp4-1(RNAi) animal showed no labeling in in situ hybridizations with a smedbmp4-1 riboprobe. Anterior, left. Scale bar: 0.1 mm.

 

Figure 2
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  		Fig. 2. A slow transformation of adult form occurs following prolonged perturbation of smedbmp4-1 signaling. Control unc-22(RNAi) animals are shown on the left, or top, of each set of images. (A) unc-22(RNAi) and smedsmad4-1(RNAi) animals were pictured following ten dsRNA treatments and 83 total days of treatment. smedbmp4-1(RNAi) and smedolloid-1(RNAi) animals were pictured following seven dsRNA treatments and 65 days of total treatment. smedolloid-1(RNAi) animals display dorsal tissue ruffling (white arrow), and smedsmad4-1 and smedbmp4-1(RNAi) animals display extra photoreceptors (yellow arrows). Anterior, left. Scale bars: 0.1 mm. (B) Magnification of fixed animals shows the extra photoreceptors in smedsmad4-1(RNAi) (126 days of RNAi) and smedbmp4-1(RNAi) (104 days of RNAi) animals. Anterior, up. (C-E) smedsmad4-1(RNAi) animals were fixed 126 days following initial dsRNA exposure and smedbmp4-1(RNAi) animals were fixed 104 days following initial dsRNA exposure. Anterior, left. Scale bars: 0.1 mm. (C,D) Animals were labeled with antibodies that recognize the photoreceptor neurons (VC-1, anti-Arrestin) and the cephalic ganglia (SYT, anti-Synaptotagmin). (D) Extra ventral nerve cords (green, vnc2) were present in a smedsmad4-1(RNAi) animal, located dorsal to the original nerve cords (red, vnc1). Animals were imaged with Zeiss Apotome-based optical sectioning, and the two sets of nerve cords false-colored. The regions shown are in the posterior of the animals. (E) In situ hybridizations with the smedbmp4-1 riboprobe. smedsmad4-1(RNAi) animals had no detectable smedbmp4-1 expression. (F) Animals were labeled with an antibody that recognizes cilia (anti-acetylated tubulin). The dorsal surface of smedsmad4-1(RNAi) animals display ventral-like cilia. The smedbmp4-1(RNAi) animals were fixed 117 days following initial exposure to dsRNA. Anterior, left. Scale bars: 0.05 mm.

 

Figure 3
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  		Fig. 3. The smedbmp4-1 pathway is needed for regeneration of a normal midline. (A) The diagram on the left shows the sites of amputations (black lines); data for trunks regenerating new heads and tails are shown. Animals were at 7 (unc-22 and smedbmp4-1 RNAi animals), 8 (smedolloid-1 and smedsmad4-1 RNAi animals) and 6 days (smedsmad4-1 prolonged RNAi) of regeneration. Yellow asterisks mark indented cephalic blastemas and red asterisks mark indented caudal blastemas. (B) Animals were labeled with the D.21.6 riboprobe that recognizes cells at the animal boundary (purple). Black arrows indicate cephalic and caudal blastema midline. The unc-22(RNAi) and smedolloid-1(RNAi) animals were at 11 days of regeneration, the smedsmad4-1(RNAi) animal was at 8 days of regeneration, and the smedbmp4-1(RNAi) animal was at 9 days of regeneration. (C) Prolonged exposure to smedsmad4-1 dsRNA completely blocked blastema formation. Animals were fed dsRNA on days 1, 5, 13 and 17 and amputated the following day. Animals were decapitated and had tails removed. Blastema formation failed in the smedsmad4-1(RNAi) animals at 6 days following amputation (middle). Outgrowths (arrows) of tissue from pre-existing tissue in a smedsmad4-1(RNAi) animal showed head-like structures at 16 days of regeneration (right). Scale bars: 0.1 mm. Anterior, left.

 

Figure 4
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  		Fig. 4. smedbmp4-1 signaling is required for blastema patterning. White lines indicate approximate boundary between pre-existing tissues and blastema. The diagram on the left shows the sites of amputations (black lines); data for trunks regenerating new heads are shown. Anterior, left. (A) Animals were labeled with an antibody that recognizes cilia (anti-acetylated tubulin). White arrows indicate aberrant cilia patterning at the midline of cephalic blastemas. The dorsal surface is shown. Animals had between 7 and 9 days of regeneration. Scale bars: 0.1mm. (B) Animals were labeled with antibodies that recognize the photoreceptor neurons (VC-1, anti-Arrestin) and the cephalic ganglia (SYT, anti-Synaptotagmin). Animals had between 7 and 8 days of regeneration. P, photoreceptors. Arrows indicate midline of cephalic ganglia and optic chiasmata.

 

Figure 5
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  		Fig. 5. Differentiation abnormalities of lateral blastemas in animals with perturbed smedbmp4-1 signaling. (A-D) Diagrams in A and C show the sites of amputation (black lines) that generate thick or thin fragments that lack bilateral symmetry. Animals were examined at 14 days (unc-22 and smedsmad4-1 RNAi animals) and 22 days (smedolloid-1 and smedbmp4-1 RNAi animals) after amputation. White arrows indicate the wounded side. (A,B) Thick fragments. (C,D) Thin fragments. (B,D) Animals were labeled with the D.21.6 riboprobe that recognizes cells at the animal boundary (purple). Anterior, left. Scale bar: 0.1 mm.

 

Figure 6
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  		Fig. 6. smedbmp4-1 is expressed in the pre-existing tissue of asymmetric fragments lacking a midline and expands towards wound sites in asymmetric fragments containing a midline. (A-D) Diagrams show a model of the smedbmp4-1-expressing midline, with black and red lines indicating the sites of amputation or injury. Arrows indicate new smedbmp4-1 expression. In situ hybridizations were performed with the smedbmp4-1 riboprobe. (A,B) Anterior, left. Thin, lateral wild-type fragments were generated that lacked a pre-existing midline. New smedbmp4-1 expression was detected in these fragments by in situ hybridization. (B) Animals were irradiated with 6,000 Rad prior to amputation. New smedbmp4-1 expression was detected 48 hours later (left), which failed to resolve to a midline pattern (right). (C) Representative animals (thick fragments) fixed and labeled using the smedbmp4-1 riboprobe, 0 and 48 hours following amputation. `0' hours refers to all animals fixed shortly (between 0 and 3 hours) after surgery. The pre-existing midline of animals is defined as a line connecting the tip of the head through the photoreceptors and the middle of the pharynx, to the tip of the tail. The left (L) and right (R) regions of smedbmp4-1 expression were determined by measuring the distance from the pre-existing midline to the end of the field of expression just above and below the pharynx. Means were taken per animal from these two measurements for both the left and right sides (red lines), and at least eight animals were scored for each time point. The graph indicates the mean ratios and standard deviations of measurements taken from wild-type animals and from animals fixed shortly after amputation (0 h), 24 hours after amputation, or 48 hours after amputation. Only animals missing their right sides were scored. The ratio of the right distance to the left distance indicated expansion towards the right, and was at each time point significantly different from the controls (P<0.0001, t-test). Furthermore, 66/73 fragments that were either in the 0 h or a 24 h or later group could be identified correctly in blind scoring. (D) Left, a wild-type animal was cut with a side nick perpendicular to the midline (red line in diagram). The nick was allowed to seal and animals were fixed 26 hours later; no obvious change in smedbmp4-1 expression was detected (n=24). Middle, a wild-type animal was cut with an internal nick parallel to the midline (red line in diagram). The nick was allowed to seal and was fixed 48 hours later; no obvious change in smedbmp4-1 expression was detected (n=15). Right, a wild-type animal was cut longitudinally (red line in diagram). 48 hours later no obvious change in smedbmp4-1 expression was detected (n=11). Scale bars: 0.1 mm. h, hours; d, day.

 

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© The Company of Biologists Ltd 2007