Cell Death in Cortical Development: How Much? Why? So What?

Although a lot more must clearly be learned about exactly how much cortical cell death occurs and what causes it, the simple fact that there seems to be a lot more death than was previously known is enough to warrant a reevaluation of our understanding of how different cortical cell types arise, particularly as has been revealed by cell lineage experiments. Lineage studies are worth singling out because they depend on the assumption that relatively little cell death occurs. By labeling cells with a heritable marker while they are dividing and then waiting until later in development or even until maturity to analyze the progeny in labeled clones, developmental cell death that occurs between labeling and analysis could have a significant impact on the results of such studies. The many lineage studies that have recently been undertaken in the nervous system (reviewed byMcConnell 1995xMcConnell, S.K. J. Neurosci. 1995; 15: 6987–6998PubMedSee all ReferencesMcConnell 1995), especially in the retina and cortex, have assumed that too little cell death occurs to have a significant role in shaping clone composition. If half or more of the cells produced in a region die during development, however, the composition of any observed labeled clone almost certainly provides an incomplete view of the cells originally generated in that clone.My colleagues and I have explored the relationship between cell death and cell lineage in the developing rodent retina (Voyvodic et al. 1995xVoyvodic, J.T, Burne, J.F, and Raff, M.C. Eur. J. Neurosci. 1995; 7: 2469–2478CrossRef | PubMed | Scopus (42)See all ReferencesVoyvodic et al. 1995). We found that the amount of cell death that normally occurs in the retina is sufficient to obscure lineage patterns by producing much more apparent clonal variability than might actually be the case (Figure 2Figure 2). Lineage studies in the retina (8xHolt, C.E, Bertsch, T.W, Ellis, H.M, and Harris, W.A. Neuron. 1988; 1: 15–26Abstract | Full Text PDF | PubMed | Scopus (410)See all References, 15xWetts, R and Fraser, S.E. Science. 1988; 239: 1142–1145CrossRef | PubMedSee all References, 13xTurner, D.L, Snyder, E.Y, and Cepko, C.L. Neuron. 1990; 4: 833–845Abstract | Full Text PDF | PubMed | Scopus (398)See all References) have interpreted the high degree of variablity seen in clone composition to indicate that there is no underlying lineage pattern. This conclusion would imply that progenitor cells remain multipotent in terms of cell identity until they stop dividing, at which time each postmitotic cell differentiates independently depending on the signals it receives within its local microenvironment. Because our measurements in the retina only dealt with postmitotic cell death, it will be interesting, in the light of the results of Blaschke et al., to apply the more sensitive ISEL-labeling technique to see whether there is even more cell death occurring among the dividing cell population in the retina as well.Figure 2Examples of Clonal Variability Due to Cell Death(A) A hypothetical retinal cell lineage that produces seven cells with four different phenotypes; in the example, a single cell is produced for three cell types, and four cells are produced for the fourth cell type (corresponding to rod photoreceptors, which are produced in relatively large numbers).(B–D) Three examples of different clones that could result from the lineage in (A) with 50% cell death occurring in all cell types except rods. Dead cells are indicated with small black circles. The resulting clones look different even though the underlying lineages are identical. Variability in the availablility of mitogenic growth factors could provide further clonal variablility independent of either cell type induction or cell survival mechanisms.View Large Image | View Hi-Res Image | Download PowerPoint SlideWhereas the results from retinal lineage studies have emphasized clonal heterogeneity and multipotential precursor cells, cortical lineage studies have resulted in more homogeneous clones that are thought to reflect restricted cell fate potential in the precursor cells. The observation that most of the cells produced in the cortex seem to die during development does not necessarily mean that any of the conclusions based on lineage studies are wrong, but it does suggest that we carefully consider how death might alter lineage interpretations.There are two major reasons why cell death is a significant problem for interpreting cell lineage studies. The first is the simple fact that the observed clones are incomplete, so that one cannot conclude for any individual clone that the observed cells are as closely related as they might appear, nor that cell types that are not observed were not produced in that clone. A clone containing only two cells, for example, could be assumed to contain sister cells that result from a single cell division in the absence of cell death, but with cell death might conceivably represent distant cousins. Similarly, a clone with only a single type of cell might tell us something about the potential of the precursor cell in the absence of cell death, but with cell death there might originally have been other cell types that did not survive.The second reason why cell death complicates lineage analysis is the fact that we do not know why cells die. If cell death is random or occurs with uniform probability, then the effects of cell death on clone composition could be dealt with on a statistical basis given sufficient measurements. For example, if death is the result of random errors in DNA replication with a 50% probability that any particular cell would die, then the lineage results could simply take that survival rate into account when calculating clone sizes and assume that except for size the patterns of lineages observed over many clones accurately reflect the patterns that were originally generated. The same argument would hold if death is due to dependence on some nonspecific survival factor that is present at close to threshold levels, such that the survival of any individual cell is simply determined by whether its particular threshold requirement is satisfied.But as soon as the survival probability for different cell types becomes nonuniform, or if the survival of one cell is affected by the survival of neighboring cells, as is the case when cells compete for limited survival factors, then incorporating cell death into a lineage analysis becomes considerably more complicated. Competition, for example, would act to select against similar cell types within a clone because the presence of a closely related competitor would tend to decrease a cell's own probability for survival. Of course, nonuniform survival probability would also affect the distribution of different cell types within clones, perhaps killing off many or all cells of some types while sparing cells of other types. Similarly, if the probabilities of survival change with time during development, then clone composition will appear to change even if the lineages originally generated are all the same. Differences in survival conditions between cell lineage analyses performed in vivo and in vitro could also lead to apparent differences in clone composition, which might have nothing to do with differences in cell potential. Finally, the possibility raised by Blaschke et al. that large scale death in neurogenesis might reflect cell-specific phenotype recognition mechanisms similar to those occurring in the thymus further complicates lineage interpretations by shifting the cell selection process from cell populations down to the level of individual cells. Even without the extreme degree of phenotypic heterogeneity produced by antigen-specific receptor gene rearrangements in the thymus, one could imagine a scenario where some significant degree of neuronal specificity could be generated by a similar combination of nonspecific cell diversification followed by phenotypic selection.What conclusions can and cannot be safely drawn from cell lineage analysis without worrying about the impact of cell death on clone composition? One can conclude, for example, that cell types seen together in mixed clones share a common ancestor. One cannot conclude that different cell types arise from divergent cell lineages based on seeing only unmixed clones, unless multiple kinds of unmixed clones are consistently seen within a single age and location; in the latter case, it is hard to imagine cell type selection mechanisms that would result in clone-specific selection unless those clones themselves are somehow phenotypically different lineages. Finally, one cannot determine when cell identity decisions are made based on clonal variability, unless the amount of variability caused by selective survival can somehow be identified and factored out.Clearly, without knowing how much cell death occurs in development or what causes it, one could speculate forever on cell selection schemes and their roles in generating any number of observed cell lineage patterns. By the same token, however, disregarding potentially large amounts of cell death when evaluating cell lineage data could lead to a similarly fanciful view of the underlying developmental mechanisms involved. Until more is known about cell death in regions like the developing cortex, it is perhaps best not to disregard selective survival as a potentially important mechanism in determining both the number and cell type of the neurons and glia that are found in the the mature nervous system.