Adult somatic stem cells (ASCs) produce descendents that differentiate to replace cells lost to physiological turnover, age, disease, and injury (Blanpain et al., 2004
; Morrison et al., 1996
; Morrison et al., 1997
; Ohlstein and Spradling, 2006
; Romanko et al., 2004
; Weissman, 2000
; Xie and Spradling, 2000
). ASCs are normally lineage restricted in vivo
such that they typically generate only the cell types found in the tissues in which they reside (Anderson et al., 2001
; Morrison, 2001
; Wagers et al., 2002
). The specification of ASC descendents to supply a defined number and type of differentiated cells depends on the function and age of the organ, and the demands imposed by physiology, disease or unexpected trauma. A detailed mechanistic understanding of how ASCs are regulated to generate distinct lineages under normal and aberrant conditions remains a central unresolved issue of developmental biology and regenerative medicine.
Temporal and spatial coordination of changes in gene expression can produce a diverse number of differentiated cell types. For example, the sequential expression of four transcription factors in the neural stem cells of Drosophila melanogaster
is critical for proper formation of the developing CNS (Grosskortenhaus et al., 2005
; Isshiki et al., 2001
), and co-expression of multiple transcription factors in the same ASC can affect lineage choices in the vertebrate retina (Hernandez et al., 2007
; Peters and Cepko, 2002
; Wang and Harris, 2005
) and hematopoietic system (Iwasaki et al., 2006
; Mansson et al., 2007
). Additionally, specific changes in gene expression are detected as the ASCs of the mammalian intestine exit their resident microenvironment (niche), cease their proliferation and begin to differentiate along distinct lineages (Blanpain et al., 2007
). In the case of tissue regeneration, ASC fate specification is likely to pose an additional level of complexity, as some ASCs may be specified toward a distinct lineage only after a major challenge to the system occurs (e.g.
, injury), instead of contributing to physiological turnover (Ito et al., 2005
The ASCs of the planarian Schmidtea mediterranea
provide an interesting model for studying both stem cell function and lineage commitment during homeostasis and regeneration. The adult flatworm contains a large population of mitotically active ASCs, known as neoblasts (Randolph, 1892
), which generate a constant supply of progeny to sustain the high rate of physiological somatic cell turnover (Pellettieri and Sánchez Alvarado, 2007
). Planarians display unique plasticity, as small fragments removed from almost anywhere in their bodies can regenerate entire animals (Morgan, 1898
), indicating that ASCs can produce all the different cell types found in the adult flatworm. Historically, the term neoblast has been used to describe the planarian stem cells based solely on their morphology (Hori, 1982
; Hyman, 1951
), and therefore, a longstanding question in planarian biology is whether all the ASCs are totipotent, or if the population is molecularly heterogeneous.
The development of cell (Newmark and Sánchez Alvarado, 2000
; Robb and Sanchez Alvarado, 2002
) and molecular (Newmark et al., 2003
; Sánchez Alvarado et al., 2002
; Zayas et al., 2005
) reagents has facilitated the study of stem cells in planarians. Planarian ASCs express conserved regulators of stem cell function in other organisms (Guo et al., 2006
; Reddien et al., 2005b
), and are widely distributed throughout the mesenchyme of the animal, yet absent in the pharynx and the region anterior to the photoreceptors. Pulse experiments with the thymidine analog BrdU have demonstrated that ASCs are initially the only cells which incorporate BrdU, but that over time the labeled progeny migrate to post-mitotic tissues (e.g.
, anterior region of the animal), and integrate into differentiated tissues (e.g.
, epidermis) (Newmark and Sánchez Alvarado, 2000
; Reddien et al., 2005b
). Exposure to γ-irradiation effectively ablates neoblasts, which prevents planarians from maintaining physiologic homeostasis and regenerating after injury (Bardeen, 1904
; Dubois, 1949
; Reddien et al., 2005a
). Two populations of irradiation-sensitive cells with neoblast morphology can be isolated using Fluorescence Activated Cell Sorting (FACS) (Hayashi et al., 2006
; Reddien et al., 2005b
), yet it is still unclear how the isolated irradiation-sensitive cells relate to those observed with BrdU labeling studies performed in vivo
. Despite significant advances, the lack of a defined panel of markers that label discrete cell types has prevented a detailed molecular and lineage characterization of the ASCs in planarians.
In this study, we defined the expression profiles of wild type and irradiated animals, and examined expression levels and spatial distribution of the most severely affected genes both in FACS-purified cells and by whole-mount in situ hybridizations. Lineage tracing experiments using a combination of BrdU and in situ hybridization revealed robust spatial and temporal regulation of gene expression and cell proliferation in both intact and regenerating animals. Our studies define a novel set of genes specifically expressed in either planarian ASCs or their descendants. Altogether, our findings establish S. mediterranea as a powerful experimental paradigm for the adult, in vivo study of the population dynamics of tissue stem cells during both normal physiological cell turnover and injury induced regeneration.