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First published online 27 August 2003
doi: 10.1242/dev.00710

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Digit regeneration is regulated by Msx1 and BMP4 in fetal mice

Division of Developmental Biology, Department of Cell and Molecular Biology, and The Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70118, USA

View larger version (54K): [in a new window]   Fig. 1. Fetal digit tip formation. (A) An Alcian Blue stained stage 11 digit (E14.5), showing the chondrogenic structure of the digit at the time of amputation. tp, terminal phalanx. (B-F, K-L) In situ hybridization of sagittal sections of neonatal digits. Left side of each image is dorsal; top is distal. (H-J) In situ hybridization of frontal sections of the E14.5 digits. The top of each image is distal. (B) Msx1 transcripts are localized to the loose connective tissue subjacent to the nail organ (n) and surrounding the dorsal region of the terminal phalanx in the P7 digit. (C) Msx2 transcripts are localized in the nail bed and nail matrix of the P7 digit. (D) Bmp4 transcripts are expressed in dorsal loose connective tissue cells beneath the nail bed in the P2 digit. (E) Ihh is expressed at the distal tip of terminal phalanx in the newborn digit. (F) Hoxc13 is expressed in the nail bed and nail matrix of the newborn digit. (G) Whole-mount in situ hybridization of Msx1 in the stage 11 autopod. (H) Msx1 is expressed in the apical mesenchymal cells surrounding the forming terminal phalanx. The line indicates the amputation level that elicits a regeneration response. Digit amputation at a level proximal to this line does not elicit a regeneration response. (I) Msx2 is expressed in the apical epidermis and in mesenchymal cells subjacent to the epidermis. (J) Bmp4 is expressed in apical mesenchymal cells in a domain similar to that of Msx1. (K) Ihh is expressed in digit tip cells, initiating endochondral ossification of the terminal phalanx (arrow). (L) The nail organ marker, Hoxc13, is expressed in the distal epidermis associated with presumptive nail tissue (arrow).

View larger version (45K): [in a new window]   Fig. 2. Digit regeneration in vivo. (A) E17.5 autopod (stage 13), ventral view. The central 3 digits, digits 2, 3 and 4, were amputated at E14.5 and analyzed 3 days later. Note the regenerated digit tips (asterisk) are shorter than a non-amputated control digit tip (arrowhead). (B-F,H-L) In situ hybridization of regenerated digit tips 4 days (B-F) and 2 days (H-L) after amputation. (B-D,H-L) Frontal sections with distal toward the top of the image. (E-F) Sagittal sections with distal toward the top and dorsal to the left of the image. Sections in B-F show expression of Msx1 in the mesenchyme surrounding terminal phalanx (B), Msx2 in the apical epidermis (C), low levels of Bmp4 in the apical mesenchyme (D), Ihh in the forming terminal phalanx (E), and Hoxc13 in the nail organ (F). (G) Proximally amputated digits fail to mount a regeneration response and are negative for the expression of Bmp4. (H-L) In situ hybridization of 2 day regenerates showing expression of Msx1 (H), Msx2 (I), Bmp4 (J), Ihh (K) and Hoxc13 (L). The expression patterns of these marker genes in regenerating digits are largely similar to those of developing digits with the exception of Msx2, which displays an expanded mesenchymal domain.

View larger version (68K): [in a new window]   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.

View larger version (63K): [in a new window]   Fig. 4. Expression patterns of Msx2 and Bmp4 in Msx1 mutant digits. (A,B) Msx2 whole-mount in situ hybridization of stage 11 wild-type (A) and Msx1 mutant (B) digits that were processed and imaged in parallel, showing an upregulation of Msx2 expression in the Msx1 mutant. (C) Frontal section of an Msx1 mutant digit showing the expansion of the Msx2 expression domain (compare with Fig. 1I). (D,E) Bmp4 whole-mount in situ hybridization of stage 11 wild-type (D) and Msx1 mutant (E) digits that were processed and imaged in parallel. Bmp4 expression is downregulated in the Msx1 mutant digit. (F) Frontal section of an Msx1 mutant digit showing compression of the Bmp4 expression domain to the apical mesenchyme (compare with Fig. 1J).

View larger version (73K): [in a new window]   Fig. 5. Effect of BMP4 and noggin on digit regeneration. (A-C) Exogenous treatment with BMP4 rescues the Msx1 regeneration defect. (A,B) Frontal section in situ hybridization with distal toward the top of the image. (C) Sagittal section in situ hybridization with distal to the top and dorsal to the left of the image. BMP4-induced mutant digit regeneration displays normal expression of Msx2 (A), Bmp4 (B) and Hoxc13 (C). (D-F) Exogenous treatment with the BMP-binding protein noggin inhibits regeneration in wild-type digits. (D-F) Frontal section in situ hybridization with distal toward the top of the image. Despite the absence of a regeneration response, stump tissues maintain expression of Msx1 (D), Msx2 (E) and Bmp4 (F).

View larger version (41K): [in a new window]   Fig. 6. Cell proliferation and digit regeneration. BrdU incorporation was studied in 2-day regenerating digits. (A-D) Frontal sections with distal toward the top of the image. (A) BrdU incorporation identifies a population of proliferating cells at the apex of a regenerating wild-type blastema. (B) The absence of a regeneration response in Msx1 mutant digits is associated with very little BrdU incorporation. (C) BrdU-labeled cells are shown in the regenerating blastema of Msx1 mutant digits treated with BMP4. (D) Noggin treatment inhibits cell proliferation in amputated wild-type digits.