The Newt Ortholog of CD59 Is Implicated in Proximodistal Identity during Amphibian Limb Regeneration  Sara Morais da Silva, Phillip B. Gates, Jeremy P. Brockes  Developmental Cell  Volume 3, Issue 4, Pages 547-555 (October 2002) DOI: 10.1016/S1534-5807(02)00288-5

Figure 1 Nucleotide and Deduced Amino Acid Sequences of Prod 1 cDNA and the Amino Acid Sequence Alignment with Mouse and Human CD59 (A) In the nucleotide sequence, the start codon is indicated in italics and an asterisk marks the stop codon. A putative polyadenylation signal (AATAAA) is underlined by a dashed line. The putative N- and C-terminal signal peptides are underlined and flank the mature peptide, which has a potential site at Asp-89 (arrow) for derivatization with a GPI anchor. Boxed letters indicate the two peptides, 683 and 684, which were used as immunogens for rabbit antibodies to Prod 1 protein. (B) Alignment of the amino acid sequence of Prod 1 with mouse and human CD59 showing the open reading frames. The mature Prod 1 protein is numbered from the N-terminal L resulting from cleavage of the signal peptide. The putative site of GPI-derivatization is D70. The residues enclosed in a red box indicate the conserved CD59/Ly-6 family motif CCXXXXCN. The ten cysteines residues that are conserved among members of the family are arrowed. Prod 1 lacks cys at residues 6 and 10, but it is interesting that the R and D residues in these locations might possibly form a salt bridge. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 2 Regulation of Prod1 Expression in Newt Tissues and Cultured Limb Cells Samples of RNA were prepared from different newt tissues or A1 cells, and analyzed by RNase protection assay with a combination of a Prod 1 probe and a normalizing probe (EF1α). Lanes 1–4, mid-bud stage blastemas exposed for 3 days in vivo to RA/DMSO (lanes 1 and 3) or DMSO vehicle (lanes 2 and 4). Lanes 5 and 6, newt hindlimb exposed to RA/DMSO (lane 5) or DMSO vehicle (lane 6). Lanes 7 and 8, proximal (lane 7) and distal (lane 8) forelimb blastemas. Lanes 9–11, upper arm (lane 9); elbow (lane 10); and hand (lane 11). Lane 12, brain; lane 13, liver; lane 14, heart; lane 15, A1 cells; lane 16, tRNA. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 3 In Situ Hybridization Analysis of Prod 1 Expression during Newt Limb Regeneration (A) Sagittal section through a forelimb stump at one day after amputation. (B) Higher magnification view of (A). Note positive signal both on myofibers (arrowheads) and mononucleated cells (arrows). (C) Sagittal section through an early blastema at 16 days after amputation. Note the absence of signal in the wound epidermis (arrow). (D) Sagittal section through a mid-bud blastema at 25 days after amputation. (E) Higher magnification of blastemal cells from the blastema shown in (D). Note that the majority of blastemal cells are positive and the absence of signal in the epidermis (arrow). (F) Sagittal section through a digit stage blastema at 40 days after amputation. Note that the expression is observed in cartilage condensations at the distal tip of the regenerate (arrow). Scale bars: (D), 200 μm; (A), (C), and (F), 100 μm; (E), 50 μm; and (B), 25 μm. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 4 Evidence for Surface Expression of Prod 1 and Release by PIPLC: Analysis of the Cellular Localization of Prod 1 Protein in Transfected Cells (A and B) Mouse PS cells were transfected with the pCIneo vector expressing Prod 1, and live cells were reacted with antibodies to peptides 683 (A) or 684 (B), followed by indirect immunofluorescence. The arrows indicate untransfected, negative PS cells. (C) Expression of Prod 1-eGFP fusion protein in transfected PS cells. (D) Quantitation of the fluorescence intensity of PS cells expressing Prod 1-eGFP, prion protein PrP (detected by indirect immunofluorescence), and ganglioside GM1 (detected by FITC-cholera toxin) before (white bar) and after (black bar) PIPLC digestion. The data are expressed as the mean and standard deviation of the fluorescence intensity measured on 40–50 random cells. Note that Prod 1 is released by PIPLC digestion. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 5 Analysis of PD Blastemal Confrontations in Culture (A) Diagram illustrating the confrontation procedure. Confrontation of a proximal and a distal blastema results in engulfment of the distal blastema by proximal cells after 5 days in culture. In situ hybridization analysis of Prod1 mRNA expression (B), and immunohistochemistry with the 683 antibody to detect the Prod1 protein (C) on sagittal sections of a proximodistal blastemal pair after engulfment. The line in (B) and (C) delimits the boundary between the proximal blastema and the distal partner. Note that Prod 1 mRNA and protein are expressed at higher levels in the engulfing proximal partner. (D–L) Engulfment behavior of blastemal confrontation cultures and inhibition by PIPLC. The time course of engulfment is shown by sequential images at 24 hr (D, G, and J), 72 hr (E, H, and K), and 120 hr (F, I, and L). (D–F) Coculture of a proximal with a distal blastema. Note the engulfment behavior by the proximal blastema indicated by the arrows which mark the front of the labeled P cells. (G–I) Coculture of two proximal blastemas. There is no engulfment. Note also that the boundary between the two proximal blastemas is stable throughout the time course (arrows). (J–L) Coculture of a proximal with a distal blastema in the presence of PIPLC. Note that the proximal blastema does not engulf the distal one (arrows). In this figure and Figure 6, the proximal blastema is labeled with a green cell tracker dye. Scale bar, 100 μm. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 6 Specific Inhibition of PD Engulfment by Antibodies to Prod 1 Peptides (A–C) A PD pair showing PD engulfment over 5 days in culture. (D–F) Coculture in the presence of affinity purified antibodies to Prod 1 at 1:50 dilution. No engulfment is observed. (G–I) Coculture with antibodies at 1:100. Engulfment is delayed at 72 hr (compare [B] and [H]) but not at 120 hr. (J–L) Coculture with antibodies at 1:200. No inhibition is observed. (M) Coculture with rabbit IgG at 20 μg/ml. No inhibition is observed at 120 hr. (N) Coculture with affinity-purified antibodies to tomoregulin at 20 μg/ml. No inhibition is observed at 120 hours. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)

Figure 7 Schematic Diagram of a Possible Role for Prod 1 in the Cell-Cell Interaction at a PD Confrontation The mature Prod 1/CD59 protein is represented as an oval anchored in the plasma membrane. It may act as a membrane receptor, but this function is masked by adhesive homodimeric interactions between molecules on adjacent cells. Because proximal cells express a higher level than distal cells, spare receptors are available to interact with ligand (cup shape) either on the same cell or on the adjacent one. It is also possible that the ligand is diffusible (morphogen-type signal) and selectively activates the proximal cell. Activation of the spare P receptors may lead to movement/engulfment, as well as proliferation of the P cell. Thus, while several variants of this model can be envisaged, the key proposal is that cells are locally activated after confrontation by a disparity in cell surface expression. Developmental Cell 2002 3, 547-555DOI: (10.1016/S1534-5807(02)00288-5)