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. 2014 Jan 9;10(1):e1003999. doi: 10.1371/journal.pgen.1003999

Figure 5. FoxD(RNAi) animals display abnormal anterior pole, head patterning and midline gene expression.

Figure 5

(A) Upper panels: animals injected with FoxD 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 FoxD dsRNA injected animals. Defects in head regeneration of FoxD(RNAi) animals were observed; probes used: brain, chat (7/7 animals showed a medially collapsed or a very small brain); intestine, mat (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) Upper panels: double FISH (smedwi-1 in green, NB.21.11e 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 (notum, 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-t-test analysis; *p<0.05, n>10. (C) FoxD dsRNA injected animals displayed defects in anterior pole gene and PCG expression at 72 hours following amputation (sFRP-1, 5/9; ndl-4, 3/4; notum, 10/11; prep, 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. (D) Single or double FISH in day seven regenerating dsRNA injected animals. Defective expression of the anterior pole gene notum (8/20 no expression, 10/20 decreased expression) and sFRP-1 (9/18 no expression, 7/18 reduced expression) is observed in FoxD(RNAi) animals. Reduced expression of the midline genes slit (6/11 severe defect, 2/11 mild defect), admp (8/10 reduced expression), and ephrin receptor 1, ephR1 (7/14 severe reduction, 3/14 mild reduction) was observed in FoxD(RNAi) 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 admp and slit FISH. Scale bars, 100 µm.