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Fig. 3. Msx1 mutant digits display a regeneration defect. (A) Stage 11 wild-type digit tips regenerate in 3-day organ culture. Note the distal outgrowth (asterisk) associated with the regeneration response. (B-F,H-L,N-R) The expression patterns of marker genes in 3-4 day cultures (B-F) or 2-day cultures (H-L,N-R) of amputated wild-type (B-F,H-L) or Msx1 mutant digits (N-R). (B-D,H-J,N-P) Frontal sections with distal toward the top of the image. (E-F,K-L,Q-R) Sagittal sections with distal toward the top and dorsal to the left of the image. (B) Msx1 is expressed in the regenerating mesenchymal cells of wild-type digit blastemas. (C) Msx2 is expressed in the basal layer of the apical epidermis and is weakly expressed in the distal digit mesenchyme of wild-type regenerates. (D) Bmp4 is expressed in the distal mesenchyme of regenerating wild-type digits. (E) Ihh is expressed in the differentiating terminal phalanx of regenerating wild-type digits. (F) Hoxc13 expression in the apical epidermis is associated with the forming nail in regenerating wild-type digits. (G) Digit amputation fails to elicit a regeneration response (asterisk) from Msx1 mutant digits after 3 days of culture. (H-L) Regenerating wild-type digits cultured for 2 days show the reformation of the digit blastema. Msx1 (H) and Bmp4 (J) are expressed distal mesenchymal cells, and Msx2 (I) is expressed in the distal mesenchyme and apical epidermis. Ihh (K) is expressed in the differentiating terminal phalanx. Hoxc13 (L) expression is associated with the distal epidermis associated with the presumptive nail organ. (M) Msx1 mutant digits amputated in vivo and analyzed 3 days post-amputation fail to mount a regeneration response. Asterisks indicate non-regenerating digit tips. (N-R) Non-regenerating Msx1 mutant digits fail to express regeneration marker genes 2 days after amputation. (N) Expression of non-functional Msx1 transcripts is detected in the distal mesenchyme. Msx2 (O), Bmp4 (P), Ihh (Q) and Hoxc13 (R) are not expressed in the mutant digit following amputation.