<i>FoxD</i> is wound-induced in the midline.

<p>(<b>A</b>) Wild-type animals were transversely amputated, fixed at six hours following wounding, and <i>FoxD</i> (purple) expression was analyzed by <i>in situ</i> hybridization. Red box in cartoon on the left shows the area imaged. All animals showed anterior and posterior <i>FoxD</i> expression at the midline domain following wounding (n>10). Animals are anterior up; ventral view. Area within dotted black box is enlarged in right panels. Scale bar, 500 µm. Right panels, scale bar, 100 µm. (<b>B</b>) Double FISH, <i>notum</i> (green) and <i>FoxD</i> (magenta) in wild-type (0 rads) and lethally irradiated animals (6,000 rads) at different time points following transverse amputation. Red box in cartoon on the left shows the area depicted. Images shown are maximal intensity projections. Images are representative of results seen in n>6 animals per panel. Dorsal view for the 72 hours time point, ventral view for all others. Dotted white line marks the approximate wound boundary. Scale bars, 100 µm. (<b>C</b>) Cartoons show injury types performed in wild-type animals. Dotted black line shows the wound site. Type of wound from left to right: parasagittal, sagittal, incision, wedge, dorsal midline puncture, lateral edge, and lateral puncture. Red box shows the region imaged below. Whole-mount <i>in situ</i> hybridization using <i>FoxD</i> (purple) RNA probe at six hours following wounding of wild-type animals. Red arrows indicate <i>FoxD</i> expression. Dotted black box is enlarged in the inset. Images are representative of results seen in >10 animals per wound type. Animals are anterior up, ventral view. Scale bars, 500 µm. Inset scale bars, 100 µm. (<b>D</b>) Double FISH, <i>FoxD</i> (magenta) and the immediate early gene <i>fos-1</i> (green) of wounded wild-type animals fixed at six hours following midline (upper panel) and lateral (lower panel) puncture injuries. Cartoon on the left shows type of wound, red box is area imaged. Dotted yellow line depicts the approximate midline, white line depicts the animal edge. Area within dotted white box is shown in the inset for <i>FoxD</i> expression. Yellow arrows point to <i>FoxD</i>-expressing cells at the midline. Images shown are maximal intensity projections. Images are representative of results seen in >10 animals per panel. Anterior is up, ventral view. Scale bars, 100 µm (<b>E</b>) Double FISH, <i>FoxD</i> (green for upper two panels, magenta all other panels) and midline, W2 genes or the muscle gene <i>collagen</i> of wild-type animals fixed six hours following transverse amputation. Percentage (mean ± SD) of <i>FoxD</i> co-expression with <i>slit</i> was 86.3±5.5% (n = 124 <i>FoxD⁺</i> cells examined), with collagen was 92.6±4.6% (n = 52 <i>FoxD⁺</i> cells examined). Red box shows the area imaged. Anterior wounds are up, ventral view. Insets show higher magnification of co-expressing cells, scale bars are 10 µm. For the double <i>FoxD</i>, <i>collagen</i> FISH, area within dotted white box is shown in the right panel. Images shown are maximal intensity projections. Images are representative of results seen in >5 animals per panel. Dotted white line marks the approximate wound boundary. Scale bars, 100 µm.</p

<i>FoxD(RNAi)</i> animals display abnormal anterior pole, head patterning and midline gene expression.

<p>(<b>A</b>) Upper panels: animals injected with <i>FoxD</i> dsRNA regenerated normally sized anterior blastemas with one eye (73/130) or smaller blastemas with no eyes (24/130) seven days following amputation. Scale bars, 200 µm. Lower panels: double FISH and immunostainings using anti-ARRESTIN (VC1) antibody in regenerating <i>FoxD</i> dsRNA injected animals. Defects in head regeneration of <i>FoxD(RNAi)</i> animals were observed; probes used: brain, <i>chat</i> (7/7 animals showed a medially collapsed or a very small brain); intestine, <i>mat</i> (3/7 animals showed some regeneration defect in the anterior blastema). Images shown are maximal intensity projections. Scale bars, 100 µm. Anterior is up, dorsal view. (<b>B</b>) Upper panels: double FISH (<i>smedwi-1</i> in green, <i>NB.21.11e</i> in magenta) and immunostaining with anti-phospho histone 3 (H3P in blue) of tail fragments fixed at 18 hours following amputation of eight weeks RNAi fed animals. Red box in cartoon on the left shows the area imaged. Lower panels: FISH (<i>notum</i>, green) and immunostaining with anti-H3P (blue) were performed at 48 hours following amputation in regenerating tail fragments of eight weeks RNAi fed animals. Anterior is up, dorsal view. Images shown are maximal intensity projections. Scale bars, 100 µm. Numbers of mitotic cells were counted and normalized by the tail area (mm²) and analyzed using a Student-<i>t</i>-test analysis; *p<0.05, n>10. (<b>C</b>) <i>FoxD</i> dsRNA injected animals displayed defects in anterior pole gene and PCG expression at 72 hours following amputation (<i>sFRP-1</i>, 5/9; <i>ndl-4</i>, 3/4; <i>notum</i>, 10/11; <i>prep</i>, 5/6). Dotted white line marks the approximate animal edge. Images shown are maximal intensity projections. Anterior is up, dorsal view. Scale bars, 50 µm. (<b>D</b>) Single or double FISH in day seven regenerating dsRNA injected animals. Defective expression of the anterior pole gene <i>notum</i> (8/20 no expression, 10/20 decreased expression) and <i>sFRP-1</i> (9/18 no expression, 7/18 reduced expression) is observed in <i>FoxD(RNAi)</i> animals. Reduced expression of the midline genes <i>slit</i> (6/11 severe defect, 2/11 mild defect), <i>admp</i> (8/10 reduced expression), and <i>ephrin receptor 1, ephR1</i> (7/14 severe reduction, 3/14 mild reduction) was observed in <i>FoxD(RNAi)</i> animals. Yellow arrows point to missing or aberrant expression. Images shown are maximal intensity projections. Anterior is up, dorsal view for all panels except for <i>admp</i> and <i>slit</i> FISH. Scale bars, 100 µm.</p

A <i>forkhead</i> Transcription Factor Is Wound-Induced at the Planarian Midline and Required for Anterior Pole Regeneration

<div><p>Planarian regeneration requires positional information to specify the identity of tissues to be replaced as well as pluripotent neoblasts capable of differentiating into new cell types. We found that wounding elicits rapid expression of a gene encoding a Forkhead-family transcription factor, <i>FoxD</i>. Wound-induced <i>FoxD</i> expression is specific to the ventral midline, is regulated by Hedgehog signaling, and is neoblast-independent. <i>FoxD</i> is subsequently expressed within a medial subpopulation of neoblasts at wounds involving head regeneration. Ultimately, <i>FoxD</i> is co-expressed with multiple anterior markers at the anterior pole. Inhibition of <i>FoxD</i> with RNA interference (RNAi) results in the failure to specify neoblasts expressing anterior markers (<i>notum</i> and <i>prep</i>) and in anterior pole formation defects. <i>FoxD(RNAi)</i> animals fail to regenerate a new midline and to properly pattern the anterior blastema, consistent with a role for the anterior pole in organizing pattern of the regenerating head. Our results suggest that wound signaling activates a <i>forkhead</i> transcription factor at the midline and, if the head is absent, <i>FoxD</i> promotes specification of neoblasts at the prior midline for anterior pole regeneration.</p></div

<i>FoxD(RNAi)</i> animals have normal expression of wound-induced genes and posterior genes.

<p>(<b>A</b>) Double FISH using <i>notum</i> (green) and <i>wnt1</i> (magenta) in trunk fragments of control and <i>FoxD</i> dsRNAi injected animals six hours following amputation. Red box in cartoon on the left shows the area imaged. Graph shows total number of cells expressing <i>notum</i>, or <i>wnt1</i> in the different RNAi conditions. Data are shown as mean± SD, n>5 animals per group. Anterior is up, ventral view. (<b>B</b>) Posterior blastemas at day seven of regeneration of <i>FoxD</i> dsRNA injected animals are shown; expression of posterior patterning gene <i>wnt11-2</i> (17/17) and the posterior pole gene <i>wnt1</i> (14/14) was normal in <i>FoxD(RNAi)</i> animals. Anterior is up, dorsal view. For all panels, images shown are maximal intensity projections. Scale bars, 50 µm.</p

Hh signaling is required for wound-induced <i>FoxD</i> expression.

<p>(<b>A</b>) Double FISH; <i>notum</i> (green) and <i>FoxD</i> (magenta) of RNAi fed animals fixed six hours following transverse amputation. Red box in cartoon shows region imaged. Images shown are maximal intensity projections. Dotted white line marks the approximate wound boundary. Anterior wounds are up, ventral view. Scale bars for all panels, 100 µm. (<b>B</b>) Graphs show number of cells expressing <i>FoxD, notum</i>, or <i>wnt1</i> in the different RNAi conditions. Data are shown as mean ± SD, and analyzed using a one-way ANOVA test; **p<0.01, ***p<0.001, n>10 animals per RNAi condition.</p

<i>FoxD</i> is expressed in anterior pole progenitors.

<p>Double FISH in wild-type head blastemas 72 hours after transverse amputation. Red box in cartoon shows the region imaged. Images shown are maximal intensity projections. Images are representative of n>5 animals per panel. Anterior is up, dorsal view. (<b>A</b>) <i>FoxD</i> (magenta) and PCGs (green). Percentages (mean ± SD) of <i>FoxD</i> cells co-expressing anterior patterning genes and anterior pole markers are as follows: 95±2% with <i>sFRP1</i>; 93±3% with <i>ndk</i>; 81±9% with <i>ndl-4</i>; 86±5% with <i>prep</i>; and 74±11% with <i>notum</i>; co-expression with the midline gene <i>admp</i> is 3±5% (n>120 <i>FoxD⁺</i> cells examined in each case). Scale bars, 100 µm. Inset shows higher magnification of co-expressing cells, scale bars are 10 µm. (<b>B</b>) <i>FoxD</i>, <i>notum</i>, and <i>prep</i> (magenta), <i>smedwi-1</i> (green). Percentage (mean ± SD) of <i>FoxD</i> cells co-expressing <i>smedwi-1</i> was 39.7±13.3% (n = 157 <i>FoxD⁺</i> cells examined); percentage of <i>notum</i> cells co-expressing <i>smedwi-1</i> was 22.9±6.1% (n = 111 <i>notum⁺</i> cells examined). Yellow arrows point to double-labeled cells. Scale bars in upper left panel, 100 µm; other panels, 10 µm.</p

<i>FoxD</i> is expressed with multiple other PCGs at the anterior pole.

<p>Wild-type intact animals were labeled in double whole-mount fluorescence <i>in situ</i> hybridization (FISH) with <i>FoxD</i> (magenta) and several anterior and midline patterning genes (green). Percentages (mean ± SD) of <i>FoxD</i> cells co-expressing anterior markers are: 56±5% with <i>sFRP1</i>; 66±9% with <i>ndk</i>, 43±9% with <i>ndl-4</i>, 72±19% with <i>prep</i>, and 68±11% with <i>notum</i>; co-expression with the midline genes <i>admp</i> is 2±4%, and <i>slit</i> is 5±8% (n>50 <i>FoxD⁺</i> cells examined in each case). Red box in cartoon on the left shows the area depicted. Animals are anterior up; dorsal view. Insets show higher magnification images of co-expressing cells. Images shown are maximal intensity projections. Images are representative of results seen in >6 animals per panel. Scale bars for all panels, 100 µm. Inset scale bars, 10 µm.</p