Large numbers of cells die by apoptosis during the development of multicellular organisms (see the 'Background' box), and many research groups are hunting down the molecular culprits responsible for clearing up the corpses. Apoptotic cells are removed by a process involving recognition and phagocytosis, followed by the induction of an active anti-inflammatory response. These events are critical for efficient corpse elimination and to prevent the leakage of potentially cytotoxic or antigenic cellular contents that could elicit an autoimmune response; defects in apoptotic cell clearance are associated with autoimmune and inflammatory diseases.

The existence of several knockouts of the same gene with very different phenotypes is puzzling, intriguing, and divides researchers in the field about how to interpret the results. Simon Brown of the University of Edinburgh, UK, urges readers to focus on the commonality of the three studies. "All three found the homozygous-null mouse to be perinatal lethal with clear evidence of a defect in cell differentiation and marked effects on tissue and organ development following the mid-gestation period," he notes. He admits that there are some major differences that draw one's attention but suspects these can be explained by differences in experimental approaches. Most researchers seem to agree that Lengeling's analysis is particularly careful, but the discrepancies between the different studies remain perplexing.

Michael Hengartner at the University of Zurich, Switzerland, is unequivocal. "Lengeling's results clearly demonstrate that Ptdsr is most certainly not a PS receptor, and probably has nothing to do with apoptotic cell recognition at all." He suspects that the source of the problem is a case of false identification during the expression-cloning of the antigen recognized by mAb 217. This is supported by the supplementary data provided by Böse et al. 2, in which mAb 217 is shown to recognize macrophages derived from their knockout mouse 2. "One wonders why the other two groups did not perform this control using their mice," notes Hengartner. Shigekazu Nagata from the Osaka University Medical School, Japan, also feels that the cloned Ptdsr gene had not been sufficiently characterized previously. "Lengeling's group now shows that Ptdsr carries an epitope that can be weakly recognized by the mAb 217. But the antibody efficiently stains even the Ptdsr-deficient cells, indicating that the antibody recognizes a molecule other than Ptdsr."

"The PS receptor story is an interesting case of how an excusable error, that can easily happen in any scientific pursuit, results in a series of published data that are guided by prejudice," says Angelika Böttger from the Ludwig-Maximilians-Universität in Munich, Germany. She is sure that the mAb 217 antibody really does inhibit the phagocytosis of apoptotic cells. "The only problem is that the Ptdsr gene does not encode the antigen for this antibody." She says that the experiment in which the Ptdsr-deficient cells are stained with the mAb 217 "should finally convince everybody that the dogma is wrong."

Other researchers remain unconvinced. "One should not rush to conclude that Ptdsr is not important for corpse removal based on the analysis of one mouse Ptdsr-knockout line," cautions Ding Xue from the University of Colorado in Boulder. "The differences in mutant phenotypes observed in the three different mouse lines, including apoptotic corpse removal, are likely due to the different genetic backgrounds of the knockout mice or differences in carrying out various assays," he says. "I think that the Lengeling group should at least analyze the mouse line from Li et al. or Kunisaki et al. before making any definitive conclusions." Xue cites numerous precedents in which different genetic backgrounds yield dramatically different mutant phenotypes "For example, caspase3-deficient mice in a C57BL/6J background are viable, but are nonviable in a 129 background. In such circumstances, one needs to be cautious about stating whether the results obtained from one mouse line are more reliable or credible than the other lines: most likely, the results from three groups were all correct in respect to the mouse lines that they examined." Lengeling's group used an isogenic C57BL/6J background, whereas the previous Ptdsr knockouts were in a mixed 129 × C57BL/6 background (see Figure 1).

Siamon Gordon, a macrophage expert at the University of Oxford, UK, feels that "the main point of this article is that people were not looking hard enough before and were jumping to conclusions." But Henson himself welcomes the different results. "The more studies we have on this molecule the more interesting it gets, and it clearly has multiple functions." Henson confides that his group has generated a fourth knockout strain and preliminary results suggest that the phenotypes differ from the other three. He admits that it appears confusing, but is confident that the data will eventually fit together." Gordon suggests that different cells may contribute to clearance in different scenarios. "Macrophages are faster and more efficient professional phagocytes than non-leukocytes, so they may be the main players during inflammation or infection." He notes that earlier studies had indicated that macrophages are not essential for apoptotic clearance during development, adding that C. elegans has no macrophages but can still clear corpses.

Many experts hope that clues will come from analysis of the Ptdsr protein in other species. Xue's group has analyzed the role of the Ptdsr gene in worms and found evidence that it is involved in removing apoptotic cells 7. But Kristin White's group at MGH-Harvard in Charlestown, USA, has studied the PS receptor in flies and came up with results that fit more with those in the Lengeling article. "We see no obvious defect in engulfment of apoptotic cells in Drosophila embryos that lack PS receptor. These animals are viable, with some subtle developmental defects," says White.

Future studies will obviously focus on functions of the Ptdsr beyond apoptotic cell clearance. Nagata hopes that the Lengeling study will trigger investigation of the 'real' function of Ptdsr during mammalian development. "These have little in common; thus Ptdsr probably has a specific cellular role that is required at multiple occasions throughout development," says Hengartner. White agrees: "Since we also see effects on development in the fly PS receptor mutant, this suggests that PS receptor has a biological function that is more general than the recognition of apoptotic cells. The use of different biological systems and approaches to dissect the role of this protein should help us to understand this more general role."

There are likely to be more surprises in store. "The Ptdsr protein carries a domain called the Jumonji C (JmjC) domain," notes Nagata, speculating that, like other proteins with this domain, Ptdsr may play a role in chromatin remodeling and the stability of heterochromatin. Edinburgh's Brown makes similar predictions and indeed, there is evidence that the Ptdsr protein may be located in the nucleus 89. Böttger, whose lab studies the PS receptor from Hydra, suggests "it could modify nuclear proteins, transcription factors or maybe proteins maintaining nuclear architecture and thus have these profound effects on differentiation during early mouse development."

Alice's adventures came to an end when she awoke from her dream. But our apoptotic adventures are likely to continue for some time as we learn more about the surprises that govern when and how cells die and who is responsible for clearing up the remains.