Stem cells depend on the surrounding microenvironment to provide signals that control their identity, self-renewal, and position and to prevent differentiation. The identification of cellular and molecular stem cell niche components has broadened our understanding of the complex interplay between stem cells and niches. In this study, we investigate the roles of integrins and their ligands in stem cell regulation in the Drosophila ovary. Our results support the model that one population of ovarian stem cells, the FSCs, produce the integrin ligand LanA, generating a critical component of their own niche (). Activation of integrin receptors expressed on the FSC surface maintains FSC position and enables FSCs to receive proliferative cues. This mechanism is not required to maintain other ovarian stem cell populations. Thus, distinct adhesion pathways regulate communication between stem cells and their niche within the same organ. The differentiating daughters of FSC divisions, prefollicle cells, also rely on integrins within the FSC to regulate their subsequent polarization and function. Therefore, integrin function within FSCs is critical for the maintenance and development of both FSCs and their progeny.
Figure 9. Model for integrin function in FSCs. (1) FSCs (yellow triangles) secrete LanA (green) into the ECM to generate a critical niche component. (2) Engagement of αPS1βPS (mew/mys, white/pink) by LanA and of αPS2βPS (if/mys, (more ...)
Emerging evidence suggests that there are two general categories of stem cell niche. Stable niches, such as the mammalian hematopoeitic stem cell niche and GSC niches in flies, can exist in the presence or absence of stem cells and provide sufficient information for cells to acquire or retain stem cell fate (Xie and Spradling, 2000
; Kai and Spradling, 2003
; Wilson and Trumpp, 2006
). In these cases, differentiated support cells perform most niche functions, including anchoring, maintenance of asymmetrical cell divisions, and the generation of factors that control proliferation and survival and prevent differentiation (Harrison and Harrison, 2006
; Wilson and Trumpp, 2006
). The architecture and position of other niches is less clearly defined, perhaps enabling the movement of stem cells across the tissue rather than fixing them in a defined location. In such “flexible” niches, there is no obvious differentiated support cell to provide anchoring function. Instead, stem cells may attach to a prominent basal lamina that surrounds the entire tissue. It is possible that stem cells in these tissues can change location in response to local signals, thus functioning where they are needed at a given point in time (Ohlstein and Spradling, 2006
We propose that FSCs reside in a dynamic niche that retains characteristics of both stable and flexible niches. Normally, the FSC niche is found halfway through the germarium on its outer surface (Margolis and Spradling, 1995
; Nystul and Spradling, 2007
) and is therefore positionally defined. Additionally, FSCs associate transiently with supporting escort cells via E-cadherin–mediated adhesion (Song and Xie, 2002
; Nystul and Spradling, 2007
). FSCs lacking E-cadherin are lost, indicating that this mechanism contributes to FSC maintenance. However, in agametic germaria, which lack germ cells and escort cells, FSCs function normally despite their drastically altered position and the absence of cellular components of the niche (Kai and Spradling, 2003
; Kirilly et al., 2005
). Thus, the FSC niche may be flexible, enabling FSCs to self-renew and produce progeny in the absence of a structurally and positionally defined niche. Our results demonstrate that integrin interactions with the basal lamina regulate FSC maintenance, positioning, and proliferation in normal germaria. Integrins also are present on the FSC basal surface in agametic germaria (Tanentzapf et al., 2000
), suggesting that this regulatory mechanism is functional even when cellular components of the niche are absent. Our results support the idea that at least one component of the niche, the integrin ligand LanA, is generated by FSCs themselves. This unique property may permit FSCs to adjust their local environment, enabling them to function in disparate locations.
Previously identified stem cell regulatory factors often are expressed or localized asymmetrically between the stem cell and its progeny cells. For example, higher levels of E-cadherin are found at the interface between ovarian GSCs and niche cells than are found between germ cells (Song et al., 2002
; Jin et al., 2008
). In contrast, integrins are expressed at apparently uniform levels in FSCs and their progeny. However, integrins are critical for FSC function and are dispensable in progeny that have already initiated differentiation. This suggests that asymmetry is generated functionally rather than by differential expression or localization.
Functional asymmetry might be achieved in two possible ways. First, FSC positioning at the anterior-most point of the developing epithelium may make it uniquely capable of receiving secreted signals produced by differentiated cells further to the anterior (Kirilly et al., 2005
). Activation of downstream pathways within the FSC, in concert with integrin-dependent signals, may promote expression of a unique set of genes that control FSC identity and behavior. Second, FSC positioning may make it uniquely dependent on integrin-mediated adhesion for its maintenance. In addition to integrin-mediated basal positioning information, differentiating prefollicle cells receive lateral and apical signals from neighboring prefollicle cells and germ cells, respectively. No current evidence suggests that FSCs receive positioning information from either germ cells or their differentiating progeny. Additionally, FSCs are only partially polarized, with clear basal domains but intermixed lateral and apical components (Tanentzapf et al., 2000
). Thus, FSCs may depend on integrins for their positioning and polarization in the absence of additional signals.
Dynamic interaction between integrins and locally produced ECM ligands is an important mechanism for controlling changes in cell migration, adhesion, and polarization that are required for development (Li et al., 2003
; Nelson and Bissell, 2006
). Laminin binding to integrin receptors both activates intracellular signaling pathways and promotes biochemical changes in the laminin network that are necessary for basement membrane formation. This process can occur in many cells simultaneously, resulting in the polarization of an entire epithelium or in individual cells to temporally and spatially control adhesion versus migration decisions (Li et al., 2003
; Medioni and Noselli, 2005
We found that FSCs lacking the integrin ligand (lanA
) or a subunit of its receptor (mew
(αPS1)) present identical FSC maintenance and proliferation phenotypes (). Additionally, the LanA produced by neighboring WT cells apparently is not sufficient to maintain mutant FSCs within the niche. Thus, reciprocal signaling between laminin and integrins may depend on cell-autonomous production of LanA by FSCs after each cell division. Local production of LanA in the immediate vicinity of the FSC may be required for efficient activation of integrin signaling cascades or to maintain a stable structure for FSC anchoring. Alternatively, FSCs may need to produce LanA during the migration or displacement steps that occur during the process of FSC replacement (Nystul and Spradling, 2007
). In all cases, WT FSCs expressing LanA would be expected to compete more effectively than lanA
mutant FSCs for positioning within the niche. These results suggest that FSCs have the capacity to generate critical components of the niche that then directs their stem cell behavior.
Studies on the roles of growth factors in FSC regulation have indicated a link between FSC positioning and proliferation control. Current data suggest that the secreted factor, Hedgehog, controls FSC proliferation rates through regulation of FSC niche size and location (Zhang and Kalderon, 2001
). Additionally, FSCs in close proximity to the source of proliferative BMP signals respond robustly. In contrast, the response of FSCs residing in their normal niche, three to five cell diameters away from the signal source, is dampened (Margolis and Spradling, 1995
; Kirilly et al., 2005
). These results are consistent with a model in which the positioning of the FSC niche at the region 2A/2B border is an important factor in FSC proliferation control.
In this study, we demonstrate critical requirements for integrins in determining FSC positioning. Loss of the β-integrin mys(βPS) or both α integrins (mew if (αPS1αPS2)) resulted in detachment of FSCs from the basal lamina and displacement to the center of the germarium. The anchoring defects were associated with reduced proliferation rates in mutant FSCs, supporting a link between FSC positioning and proliferation control. However, proliferation rates also were drastically reduced in FSCs lacking mew(αPS1) or lanA, which remained properly localized. Therefore, changes in FSC positioning are not sufficient to explain the reduced proliferation rates in all integrin mutants.
These observations suggest that integrins independently regulate FSC positioning and proliferation. Whereas our data support the idea that FSC positioning depends on integrin-mediated adhesion to the basal lamina, the integrin-dependent mechanisms that control FSC proliferation are unclear. LanA and/or integrins may participate in niche formation and maintenance, perhaps cooperating with Hh signals (Zhang and Kalderon, 2001
). Integrins also may modify the FSC response to growth factor signals, a mechanism that is well documented in mammalian cultured cells (Lee and Juliano, 2004
). Finally, activation of integrins by LanA and/or other ligands may initiate signaling cascades that regulate proliferation independently of other signals. Further analysis will be required to determine the precise molecular role of integrins in FSC proliferation control.
Our results demonstrate critical roles for integrins in regulating epithelial stem cells. Integrins may play similar roles in other epithelia, such as the mammalian skin and intestine, where stem cell adhesion to the basal lamina is thought to anchor the stem cell to its niche, enabling it to receive signals that control differentiation and proliferation. In these tissues, like the fly ovary, integrin function also is critical for proper development of stem cell progeny. FSCs lacking integrin function produced severely defective progeny, but mutation within differentiating progeny cells had little or no effect. This suggests that genetic alteration within stem cell populations has more severe defects on tissue development than identical mutations in differentiated cells within the same tissue. These results may have important implications for how mutation of critical genes within stem cell populations affect tissue development and health.