Multicolor lineage tracing methods and intestinal tumors
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The generation of chimeras, which is now a standard technology for producing gene modified mutant mice, was originally developed as a tool for developmental biology. However, the application of conventional single marker chimeric mice for developmental study was initially limited. This situation has been dramatically changed by development of multicolor chimeric mice using various kinds of fluorescent proteins. Now using our technology, up to ten different clones could be distinguished by their colors, which enable us to perform more accurate statistical analyses and lineage tracing experiments than by conventional methods. This method could be applied to visualize not only cell turnover of normal stem cells but also cancer development of live tissues in vivo. In the present review, we will discuss how these methods have been developed and what questions they are now answering by mainly focusing on intestinal stem cells and intestinal tumors.
KeywordsChimeras Mosaic Intestinal stem cells Lineage tracing Intestinal tumors
Developmental studies by generating chimeras
The first artificial method to create mammalian chimeras was developed by Tarkowski in 1961 [2, 3]. He removed zona pellucida from two early embryos and mechanically fused them together. Interestingly, when fused embryo is transferred to the uterus of a surrogate mother, it starts developing as a single large embryo, but is born at a normal size . If two lineages of cells that compose chimeric mice could be distinguished, the mice could be used for lineage tracing experiments. Later, Gardner developed another method to generate mammalian chimeras. He isolated cells from inner cell mass of blastocysts, and transferred to the blastocyst cavity of another embryo. The injected blastocysts developed as chimeras [5, 6]. Evans and Martin developed the method to culture cells from inner cell mass in vitro, and established embryonic stem (ES) cells [7, 8]. This method, blastocyst injection, is now a standard method to generate knockout mice.
Another line of generating chimeras is via methods that make use of the X chromosome inactivation or the genome imprinting of [9–11]. In this method, a marker gene, such as LacZ is put into the genome locus susceptible to gene inactivation. During the early embryonic stage, one of the two alleles is inactivated at random in each cell. In heterozygous mice, one of the two alleles has LacZ gene. As a result of gene inactivation, LacZ positive/negative two-color chimeras are formed, which could be used for lineage tracing [9–11].
Multicolor lineage tracing methods and their application to study of intestinal epithelial cells
To search for tissues that are generated by monoclonal progenitors, we analyzed various tissues from adult tetrachimeric mice . In this experiment, monoclonal tissues were almost exclusively observed in endodermal epithelial and acinar tissues. Mesodermal and ectodermal tissues are generally polyclonal in origin (our unpublished findings).
By expressing an inducible form of cre, CreER and its mutant CreERT2, which is activated by tamoxifen but not endogenous estrogen [29–32] under tissue specific promoter, multicolor chimeras can be introduced in a time and space specific manner even in the tissues of interest. Of course, the system can be used for lineage tracing method . It is not only a very powerful tool to follow the fate of stem cell-derived cells, but it adds the important information regarding clonality to the conventional lineage tracing method [26–28].
Two different models of intestinal stem cells
Basically, Lgr5 strong positive CBCs are cycling and Bmi1 strong positive cells are slow growing or resting within crypts. However, when Lgr5 positive CBCs are ablated, Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells and Bmi1-expressing cells give rise to Lgr5-expressing cells, suggesting that they can compensate with each other . If all the intestinal stem cells are cycling, they are likely to be very susceptible to DNA damage and could thus easily acquire mutations that can lead to cancers. However, human epithelial malignant tumors are extremely rare in small intestine, suggesting that there is a kind of safety system to protect against tumors in small intestine. In other tissues such as hematopoietic tissue in bone marrow and hair follicle, it has been established that long-term and short-term stem cells have distinct roles in maintaining the tissue. Short-term stem cells are cycling and mainly supply differentiated tissue cells but are short-lived. On the other hand, long-term stem cells are usually resting or slow growing, and occasionally enter the cell cycle to supply short-term stem cells . In this model, long-term stem cells are protected from DNA damage. Short-term stem cells are susceptible for DNA damage but they are short-lived and therefore they would not get cancers unless they are immortalized. From the past reports, Lgr5 positive CBCs that are cycling have the characteristics of short-term stem cells . On the other hand, Bmi1 positive stem cells tend to locate at position +4 and are slow growing, matching with the characteristics of long-term stem cells. However, as described, these two populations are in part overlapping, and at least Bmi1 positive stem cells can generate Lgr5-positive CBCs, making interpretation of the data complicated. Therefore, it is not clear whether Lgr5 positive CBCs are short-term stem cells with a capacity to generate long-term stem cells, or whether they all have a capacity to be long-term stem cells from the beginning. To further investigate the issue, multicolor lineage tracing experiments have been done.
Clonal analysis of intestinal stem cells
As described above, it has been shown by chimera analysis that epithelial cells within each crypt originate from a single progenitor during development. This raises a question as to when the single cell progenitor appears during fetal development. Interestingly, it was reported that epithelial cells in neonatal crypts are polyclonal by chimeric analyses, and gradually acquire monoclonality within the first 10–14 days [42, 43]. This means that adult type stem cells appear in the crypts during the neonatal period. Because by germ line chimeras, it is not possible to control the timing of generation of chimeras. However, to accurately examine the timing of generation of stem cells during development, inducible chimeras are required. The methods for inducible chimeras via the cre-loxp system had already been developed [24, 25]. We and others improved the methods so that they could be used for all the tissues in the mammalian body [26–28, 38], and applied the method to analyses of maintenance of intestinal progenitor cells in the crypts. In the experiments, crypt epithelial cells within one crypt are initially labeled with multiple colors; however, they gradually acquired monoclonality after 1 month (our unpublished data) . To explain the results, two models were proposed. One is that each crypt has one long-term stem cell and multiple short-term stem cells, and that a hierarchy exists between stem cells. Another model is that all the Lgr5 positive CBCs have the capacity as long-term stem cells, but constantly compete for limited space in the niche, and only a single clone survives in the long term (neutral drift model) . However, the study hypothesized that all the Lgr5 positive CBCs (approximately 15 cells per crypt) possess equal ability as long-term stem cells, and competition occurs between these equal stem cells, one of which survives. Now, it has been widely accepted that there exist two types of stem cells in crypts as described above [35, 45], and that Bmi1 positive stem cells can generate Lgr5 positive stem cells . Taking this into consideration, the model would not be so simple. These issues have not reached consensus and debate is currently ongoing.
Maintenance of crypt number by crypt fission
In vitro culture of intestinal epithelial cells
Wnt signaling pathway and cancer development
APC is an inhibitor of the Wnt signaling pathway. Mutations in the APC gene cause both genetic and sporadic adenomas. Familial adenomatous polyposis (FAP) is an inherited disease in which numerous polyps are formed mainly in epithelium of the colon, due to mutations of the APC gene [52–54]. It has been shown that APC inhibits transformation of colon epithelial cells [55, 56].
Lgr5 and its homologues are known to bind to Wnt receptors and mediate Wnt signaling pathway . It has also been shown that Lgr5 activates the Wnt signaling pathway by competing with Dkk1, the inhibitor of the canonical Wnt signaling pathway, and inhibits internalization of the LRP co-receptor [58, 59]. R-spondin1 is a ligand for Lgr4 and Lgr5 and thus activates the Wnt signaling pathway [60, 61]. Interestingly, the whole genome sequencing of human colon tumors has identified recurrent abnormal fusion genes involving R-spondin2 and R-spondin3 that enhance the Wnt signal, raising the possibility that it could be a new target for cancer therapy .
The mechanism of how activation of the Wnt signaling pathway immortalizes cancer cells, or cancer stem cells has not been clear, but it was shown that activation of the Wnt/β catenin pathway activates telomerase activity , as the accumulation of β catenin stabilizes telomerase [64–66, 67].
Clonal analyses of the origin of intestinal tumors
Clonal origin of tumors has been one of the most important themes in cancer biology. Chimera analyses should be very useful for these studies. Basically it has been imagined that, regardless of whether they are benign or malignant, tumors develop from a single cell that undergoes oncogene mutations. To examine this, the clonal origin of tumors were studied by using naturally occurring human chimeras  or by analyzing glucose-6-phosphate dehydrogenase (G6PD). In these studies, the clonal origin of sporadic tumors was studied, revealing that the origin of tumors such as chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), carcinomas in the cervix, and leiomyoma are monoclonal [68–71]. Moreover, by using experimental models, research has found that the origin of mouse colon tumors generated by azoxymethane  or DMH  is monoclonal.
However, regarding benign tumors, there have been conflicting reports. In the case of hereditary neurofibromatosis, both benign and malignant neural tumors are generated. By chimera analysis with G6PD, the clonal origin of benign (neurofibroma, neuroma) and malignant (primary neurofibrosarcoma, hereditary neurofibrosarcoma) tumors was examined. Interestingly, benign tumors were polyclonal and malignant tumors were monoclonal in origin .
In the case of human colon tumors, it is well known that a benign adenoma is formed first, followed by growth of adenocarcinoma from a part of the adenoma. Multiple genes are known to involved in carcinogenesis of the colon, including APC, ras, DCC, p53. The multistep hit model involving these genes has been proposed and is well established. The APC gene mutation by itself generates benign adenomas. However, activation of proliferation by Wnt activation leads to additional mutations in essential genes for colon carcinogenesis, and generates cancers.
In 1983, Hsu et al.  analyzed adenomas in Gardner syndrome patients by using genome imprinting of the G6PD marker, and reported that they are generally polyclonal in origin. On the other hand, in 1987, Vogelstein  utilized RFLPs (restriction fragment length polymorphism) for analyzing the clonal origin of human colon tumors (adenoma and adenocarcinoma), and reported that both adenomas and adenocarcinomas are monoclonal in origin. In 1996, Novelli et al.  analyzed adenomas in an FAP patient with XY/XO chimerism, and reported that they are generally polyclonal independent of their size. The mechanism of forming polyclonal tumors is not clearly understood. It was proposed in these reports that in FAP models, multiple adjacent polyps, each of which is monoclonal, form a single tumor by “collision”, resulting in tumors in which multiple clones participate [72, 77]. However, it has not been made clear if it is also the case with sporadic adenomas without a germ line mutation in APC. One difficulty of the analysis is that we don’t yet have a good mouse model for sporadic adenomas.
In this review, we have presented an overview of how chimeric analyses were originally developed, as well as how new methods using multicolor chimeras were applied to studies of tissue specific stem cell maintenance and the development of cancers. Newer technologies are now helping to expand research on these fundamental questions. They should be especially powerful when combined with in vitro organ cultures, and time lapse imaging of live animals, which at this time are not easy to perform, but will be possible with future technical developments.
The authors thank members of the Department of Stem Cell Pathology, Kansai Medical University for their helpful discussion. We acknowledge financial support from the following sources: Funding Program for Next Generation World-Leading Researchers, The Mochida Memorial Foundation, The Naito Memorial Foundation, The Cell Science Research Foundation, The Uehara Memorial Foundation, The Mitsubishi Foundation and The Yasuda Memorial Foundation to H.U.
Conflict of interest
The authors declare that they have no conflict of interest.