Catherine Legraverend

S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage

During the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP‐expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP‐expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b‐EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from β‐actin‐GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP‐expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP‐expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment.

Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice

S100B, the EF‐hand Ca++‐binding protein with gliotrophic and neurotrophic properties implicated in the pathogenesis of Alzheimers disease, is coined as a glial marker, despite its documented presence in rodent brain neurons. We have generated a transgenic mouse whose EGFP reporter, controlled by the −1669/+3106 sequence of the murine S100B gene, allows the direct microscopic observation of most S100B‐expressing cells in the central nervous system (CNS). From embryonic day 13 onward, EGFP expression was targeted to selected neuroepithelial, glial, and neuronal cells, indicating that cell‐specific expression of S100B is regulated at the transcriptional level during development. In adult mice, the highest level of EGFP expression was found in ependymocytes; astrocytes; and spinal, medullar, pontine, and deep cerebellar S100B neurons. Our results, thus, agree with earlier reports suggesting that S100B is not a CNS glial‐specific marker. In addition, we detected EGFP and S100B in forebrain neurons previously thought not to express S100B in the mouse, including neurons of primary motor and somatosensory neocortical areas, the ventral pallidum and prerubral field. Another interesting finding was the selected EGFP targeting to neonatal S100B oligodendrocytes and adult NG2 progenitors as opposed to mature S100B oligodendrocytes. This finding suggests that, except for oligodendrocytes at the last stage of myelin maturation, the −1669/+3106 sequence of the S100B gene is a useful reagent for driving expression of transgenes in most S100B‐expressing cells of mouse brain. J. Comp. Neurol. 457:404–419, 2003.

DCAMKL-1 Expression Identifies Tuft Cells Rather Than Stem Cells in the Adult Mouse Intestinal Epithelium

In an editorial of the last issue of Gastroenterology, Montgomery and Shivdasani comment on the known markers of mammalian intestinal epithelial stem cells. We wish to caution that staining for doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL-1), one of the putative stem cell markers mentioned in this editorial, is a highly specific and robust marker of postmitotic, differentiated, tuft cells, a minority cell lineage of the intestinal epithelium, rather than a marker for intestinal epithelial stem cells. This is important since candidate markers of intestinal stem cell are scarce and DCAMKL-1 might be especially attractive to researchers because of the availability of good antibodies, which is not the case for other, functionally validated, markers, such as Lgr5.

“The Immortal DNA Strand”: Difficult to Digest?

The “immortal DNA strand” hypothesis was originally formulated by Cairns in 1975 and proposed as a mechanism to protect the genome of tissues with high turnover, such as the intestinal epithelium and the skin, from accumulating mutations occurring during DNA replication. Cairns proposed that, at one point during development, past the phase of expansion of the stem cell population, stem cells switch from a symmetric to an asymmetric mode of cell division. During each subsequent asymmetric division, one of the two template DNA strands of each chromosome (the “stem” template) is selectively transmitted to the “stem” daughter cell, whereas mutations accruing during replication will be passed on to the short-lived “non-stem” daughter cell, together with the “non-stem” template (Cairns, 1975xCairns, J. Nature. 1975; 255: 197–200Crossref | PubMed | Scopus (884)See all ReferencesCairns, 1975). Over the past decade, a lot of effort has been put into addressing the fundamental tenet of the Cairns hypothesis, i.e., whether or not asymmetric segregation of all chromosomes (ASAC) occurs in tissues with high turnover. The most frequently used experimental approach involves labeling the “stem” template DNA strand with [3H] thymidine or BrdU when conditions are conducive to symmetric stem cell divisions (yielding two stem cells), such as during periods of rapid growth or injury-induced repair, and then monitoring for evidence of selective retention of a labeled parent strand by long-lived daughter cells. Despite indirect evidence in support of the Cairns hypothesis obtained in a variety of tissues using this type of approach (reviewed in Lansdorp, 2007xLansdorp, P.M. Cell. 2007; 129: 1244–1247Abstract | Full Text | Full Text PDF | PubMed | Scopus (117)See all References, Rando, 2007xRando, T.A. Cell. 2007; 129: 1239–1243Abstract | Full Text | Full Text PDF | PubMed | Scopus (105)See all References), the idea has remained controversial.We therefore read with interest in a recent issue of Cell Stem Cell the study from Quyn et al. (2010)xQuyn, A.J., Appleton, P.L., Carey, F.A., Steele, R.J., Barker, N., Clevers, H., Ridgway, R.A., Sansom, O.J., and Nathke, I.S. Cell Stem Cell. 2010; 6: 175–181Abstract | Full Text | Full Text PDF | PubMed | Scopus (132)See all ReferencesQuyn et al. (2010), who used a labeling approach as part of their investigation of the orientation of cell division in the intestinal epithelium. The intestinal epithelium is probably the best candidate site for ASAC for several reasons: It is the most rapidly renewing tissue in the body; it is regenerated by long-lived multipotent stem cells (Barker et al., 2007xBarker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. Nature. 2007; 449: 1003–1007Crossref | PubMed | Scopus (1810)See all References, Sangiorgi and Capecchi, 2008xSangiorgi, E. and Capecchi, M.R. Nat. Genet. 2008; 40: 915–920Crossref | PubMed | Scopus (577)See all References); and no quiescent intestinal stem cells, which could play a role of “guardian of the genome,” have been identified so far. Using DNA labeling with a nucleotide analog during postirradiation crypt regeneration, as in previous studies, Quyn et al. (2010)xQuyn, A.J., Appleton, P.L., Carey, F.A., Steele, R.J., Barker, N., Clevers, H., Ridgway, R.A., Sansom, O.J., and Nathke, I.S. Cell Stem Cell. 2010; 6: 175–181Abstract | Full Text | Full Text PDF | PubMed | Scopus (132)See all ReferencesQuyn et al. (2010) reported a distribution of label-retaining cells (LRCs), along the mouse small-intestine crypt axis, similar to that previously published by Potten et al. (2002)xPotten, C.S., Owen, G., and Booth, D. J. Cell Sci. 2002; 115: 2381–2388PubMedSee all ReferencesPotten et al. (2002), thus providing support for the Cairns hypothesis. Of note is the discrepancy in the frequency of LRCs segregating chromosomes asymmetrically reported by Potten et al. and Quyn et al. Using a second BrdU labeling assay to monitor loss of the newly synthesized DNA strands from LRCs (label-loss-at-the-second- division assay), Potten et al. reported that nearly all LRCs segregate chromosomes asymmetrically, whereas at least 40% of mitotic LRCs did not in the Quyn study.To what extent do the studies by Potten et al. (2002)xPotten, C.S., Owen, G., and Booth, D. J. Cell Sci. 2002; 115: 2381–2388PubMedSee all ReferencesPotten et al. (2002) and Quyn et al. (2010)xQuyn, A.J., Appleton, P.L., Carey, F.A., Steele, R.J., Barker, N., Clevers, H., Ridgway, R.A., Sansom, O.J., and Nathke, I.S. Cell Stem Cell. 2010; 6: 175–181Abstract | Full Text | Full Text PDF | PubMed | Scopus (132)See all ReferencesQuyn et al. (2010) validate the “immortal strand” concept? In our view, outstanding questions remain. Most notably, the possibility exists that the results of both studies might have been affected by the injury protocol used in their experiments and the cellular response to injury. However, this point has been at least partly addressed by a recent study from Falconer et al. (2010)xFalconer, E., Chavez, E.A., Henderson, A., Poon, S.S., McKinney, S., Brown, L., Huntsman, D.G., and Lansdorp, P.M. Nature. 2010; 463: 93–97Crossref | PubMed | Scopus (61)See all ReferencesFalconer et al. (2010) in which DNA strand distribution between stem and nonstem daughter cells in mouse colon sections was analyzed without prior irradiation. These authors observed a higher frequency of daughter cell pairs with extreme asymmetry than would be predicted by simulated random segregation, which they interpreted as evidence for nonrandom segregation of chromatids. However, in our view, the fact that 100% asymmetry (ASAC) was never observed may in fact argue against the Cairns hypothesis.Could the long-term label retention in the stem cell compartment observed by Potten et al. and Quyn et al., and the asymmetric segregation seen by Falconer et al., reflect the asymmetric segregation of a unique subset of chromosomes? If so, Cairnss original underlying hypothesis (1975) about protection against the consequences of accumulating mutations would no longer hold, and the physiological role of such asymmetry would be entirely unclear. One way of investigating this possibility might be to combine chromosome orientation fluorescent in situ hybridization (CO-FISH) with composites of chromosome-specific probes. Alternative experimental approaches could perhaps also avoid the concerns about prelesioning of the tissue that are inherent to the standard label retention assay. For example, an inevitable consequence of ASAC after labeling of cells with thymidine analogs is the generation of unlabelled cells after a chase period corresponding to two cell divisions (label loss at the second division). In view of the reportedly high proportion of intestinal epithelial stem cells transiting through the S phase under steady state conditions (Barker et al., 2007xBarker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. Nature. 2007; 449: 1003–1007Crossref | PubMed | Scopus (1810)See all ReferencesBarker et al., 2007), monitoring the proportion of cells with label loss at the second divison might be an attractive alternative to the label retention assay.

Hierarchy and plasticity in the crypt: back to the drawing board

The prevailing concept in the field of stem cell research is that of a multipotent self-renewing cell, positioned at the origin of a hierarchical tree of branching specificities, increasing maturity and decreasing self-renewal ability. In the epithelium of the small intestine, until very recently, the supra-Paneth crypt base columnar (CBC) cell position +4 (cp4) (counting from the bottom of the crypt) was widely assumed to be the preferred position of multipotent stem cells 1, 2. Yet electron microscopy, as well as autoradiography and lineage tracing studies, supported the presence of undifferentiated 3, actively cycling 4, multipotent CBC stem cells located between Paneth cells in the crypt 5, 6, 7. Based on the results of expression and lineage studies with Lgr5-EGFP-IRES-CreERT2 knock-in mice and Rosa26-LacZ reporter mice, it was possible to show that multipotent CBC cells expressing the Lgr5 orphan receptor are present throughout the gastro-intestinal tract 7. But they are not alone. Another recent lineage study revealed the existence of multipotent, self-renewing Lgr5− CBC cells expressing the Bmi1 proto-oncogene and preferentially located above the highest Paneth cell 8. This discovery brought the cp4 model back under the spotlight, and subsequent expression studies revealed a partial overlap between the Lgr5+ and Bmi1+ CBC cell populations 9. In a recent issue of Nature, Huan Tian and colleagues now tackle the issue of their contribution to the turnover of the intestinal epithelium 10.

Random chromosome segregation in mouse intestinal epithelial stem cells

The mammalian intestinal epithelium is endowed with a high cell turnover sustained by a few stem cells located in the bottoms of millions of crypts. Until recently, it was generally assumed that the extreme sensitivity to DNA damaging agents leading to cell death and the asymmetric mode of chromosome segregation of intestinal epithelial stem cells prevented the illicit survival of mutated stem cells and guarded against mistakes leading to aneuploidy and neoplastic transformation. Recent evidence points instead to a pool of mutipotent self-renewing stem cells capable of repairing DNA by homologous recombination significantly more efficiently than other crypt cells. Furthermore, the equilibrium between cell division and differentiation is achieved at the level of the cell population obeying to a random mode of chromosome segregation and a predominantly symmetric mode of cell division. This review summarizes the experimental findings on the mode of cell division adopted by intestinal epithelial stem cells.