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First published online 17 August 2005
doi: 10.1242/dev.01976

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Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

Nadia Mercader^1,*, Elly M. Tanaka² and Miguel Torres^1,

¹ Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, E-28049 Madrid, Spain
² Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany

View larger version (112K): [in a new window]   Fig. 1. Meis expression and regulation is conserved during axolotl embryogenesis. (A-D) Whole-mount in situ hybridization of late tail bud (A, lateral view) and prehatching (C, dorsal view) axolotl embryos, compared with E9.5 (B) and E10.5 (D) murine embryos, showing Meis2 expression in the head. (E-H) Meis2 in situ hybridization (E,F) and immunohistochemistry using Meis-a antibody (G,H) on adjacent stage (St) 41 sagittal hindbrain (E,G) and eye sections (F,H). (I-L,N-Q) Analysis of Meis expression during axolotl limb development. Meis-a immunohistochemistry (I-L) and in situ hybridization with a Meis2 riboprobe (N-Q) on adjacent cryosections from St36 (I,N), St40 (J,O) and St43 (K,L,P,Q) limb buds. (L,Q) Limb bud sections from a St43 axolotl larva treated with 0.12 mM retinoic acid (RA) for 22 hours. Arrowheads indicate the distal limit of nuclear Meis expression in the limb bud. (M) Anti-Pbx1/2/3/4 immunohistochemistry on a St42 limb section. Arrowhead indicates the limit between proximal high and distal low nuclear Pbx expression. (R) Negative control for immunohistochemical stainings. hb, hindbrain; mb, midbrain; R1, rhombomere 1; R2, rhombomere 2; tel, telencephalon.

View larger version (89K): [in a new window]   Fig. 2. Meis expression and regulation by RA during limb regeneration. (A) Comparative results of quantitative RT-PCR amplification of Meis1, Meis2 and Hoxa13 from proximal (P), distal (D) and RA-treated distal (D+RA) medium bud blastema cDNA. (B) Western blot analysis of proximal (P), distal (D) and RA-treated distal (D+RA) blastemas. LC, loading control. RA treatment in A and B was maintained for 3 days before analysis. (C-H) Immunohistochemical detection of Meis-a (red) and nuclei (green) are shown on cryosections of distal blastemas at 4 days post-amputation (C,F), or distal blastemas 3 (D,G) and 6 (E,H) days after RA injection. (F-H) Higher magnification of boxed regions in C-E. (I-K) Hoxa13 in situ hybridization of blastemas treated as in C-E. Bracket in K indicates the distal blastema domain in which Hoxa13 expression is reactivated. (L-N) Anti-Pbx1/2/3/4 immunohistochemistry on proximal (L), distal (M) and RA-treated distal (N) medium bud blastemas, revealing nuclear Pbx localization throughout the blastema.

View larger version (82K): [in a new window]   Fig. 3. In vivo fate mapping of Meis-overexpressing cells in limb regenerates. (A) Constructs used for electroporation. (B,C) Immunohistochemistry on blastema sections 1 (B) and 6 (C) days after electroporation; Meis (red), GFP (green), nuclei (blue). (D-I) Time-lapse analysis of electroporated cells during proximal limb regeneration. Early bud mid-humerus blastemas electroporated with GFP (D-F) or Meis1+Pbx1+GFP plasmids (G-I) at different times post-electroporation. White lines indicate the amputation plane; white arrows, the limit between mesenchyme and the ectodermal cap; black arrow in H, the distal-most position of electroporated cells. (J,K) Diagram of the relative contribution of electroporated cells to the PD segments (indicated at left). (J) Proportions relative to the total number of cells scored in each condition; (K) proportions relative to the total number of cells observed in control limbs. The construct combinations used are indicated above. The solid red line indicates the percentage of electroporated cells contributing to regions proximal to mid-zeugopod in controls; the dotted red line indicates this percentage in different experimental conditions. dz, distal zeugopod; pz, proximal zeugopod; ds, distal stylopod.

View larger version (65K): [in a new window]   Fig. 4. Meis overexpression induces distal-to-proximal relocation of blastema cells. (A,E) Mid-humerus medium bud blastemas 1 day after focal electroporation of EGFP (A) or Meis2+Pbx1+EGFP (E). (B,F) The same limbs after complete regeneration. (C,G) Distal early bud blastemas one day after bulk electroporation with EGFP or EGFP+Meis1a. (D,H) The same limbs after complete regeneration. (I) Proportion of limbs showing GFP-positive cells in the different PD limb segments after focal electroporation of EGFP or Meis2±Pbx1+EGFP. (J) Percentage of GFP-positive cells in each PD segment of the limbs shown in I. (K) Percentage of GFP-positive cells within the different PD segments after focal electroporation of EGFP or Meis2±Pbx1+EGFP in the distal region of a late-bud proximal blastema.

View larger version (117K): [in a new window]   Fig. 5. Anti-Meis1 and anti-Meis2 morpholinos specifically block axolotl Meis1 and Meis2 translation. Meis1 or Meis2 constructs were co-electroporated together with anti-Meis1 or anti-Meis2 fluorescein-tagged MOs into the neural tube. Sagittal neural tube sections show MO-positive cells in green (A,D,G,J), Meis in red (B,E,H,K), and both signals merged with nuclear staining in blue (C,F,I,L). Arrowheads indicate Meis-positive cells. When Meis1 MO was co-electroporated with the Meis1 expression plasmid, no Meis1 protein expression was detected in MO-loaded cells (A-C). Instead, when Meis1 MO was co-electroporated with the Meis2 expression plasmid, MO and Meis2 protein signals were found in the same cells (D-F). Reciprocally, when Meis2 MO was co-electroporated with the Meis1 expression plasmid, MO and Meis2 protein signals were found in the same cells (G-I). Instead, when Meis2 MOs were co-electroporated with Meis2 expression plasmid, no Meis2 protein expression was detected in MO-loaded cells (J-L). These results indicate the effectiveness and specificity of the Meis1 and Meis2 MOs.

View larger version (46K): [in a new window]   Fig. 6. Blockade of Meis gene function inhibits RA-induced blastema proximalization. (A,B) Longitudinal sections through distal limb blastemas, 30 hours post-electroporation with rhodamine-coupled control MO (A) or fluorescein-anti-Meis1+Meis2 MOs (B). (C) Dorsal view of an axolotl amputated bilaterally at mid-carpal level, RA-treated, and electroporated with Meis1+Meis2 MOs (right) and control MO (left). (D) Skeletal preparations of forelimbs from a single animal regenerated after RA treatment and bilateral electroporation with control MO. (E) Skeletal preparations of forelimbs regenerated after RA treatment and electroporated with control MO (left) and Meis1+Meis2 MOs (right). Each pair is from a single animal. Numbers beneath forelimbs indicate duplication indexes. S, complete extra stylopod; s, partial extra stylopod; sh, extra shoulder girdle; Z, complete extra zeugopod; z, partial extra zeugopod.

View larger version (33K): [in a new window]   Fig. 7. Effect of Meis gene function blockade on RA-induced limb duplication. Graphs indicate the number of forelimb pairs (y axis) for the different asymmetry index values (x axis). (A) Bilateral control MO electroporation (repeated in B for reference), (C) Meis1 MO (left) and control MO (right), (E) Meis2 MO (left) and control MO (right), (D) Meis1+Meis2 MO (left) and control MO (right), (F) control MO (left) and Meis1+Meis2 MO (right). L, left; R, right; LDI, left duplication index; RDI, right duplication index.