Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells

Many adult tissues are maintained by resident stem cells that elevate their proliferation in response to injury. The regulatory mechanisms underlying regenerative proliferation are still poorly understood. Here we show that injury induces Hedgehog (Hh) signaling in enteroblasts (EBs) to promote intestinal stem cell (ISC) proliferation in Drosophila melanogaster adult midgut. Elevated Hh signaling by patched (ptc) mutations drove ISC proliferation noncell autonomously. Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation. Hh signaling acted in EBs to regulate the production of Upd2, which activated the JAK–STAT pathway to promote ISC proliferation. Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation. Hence, our study uncovers a JNK–Hh–JAK–STAT signaling axis in the regulation of regenerative stem cell proliferation.     

FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools

The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1(+)/Lim2(+) neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.     

Hippo signaling in mammalian stem cells

Over the past decade, the Hippo signaling cascade has been linked to organ size regulation in mammals. Indeed, modulation of the Hippo pathway can have potent effects on cellular proliferation and/or apoptosis and a deregulation of the pathway often leads to tumor development. Importantly, emerging evidence indicates that the Hippo pathway can modulate its effects on tissue size by the regulation of stem and progenitor cell activity. This role has recently been associated with the central position of the pathway in sensing spatiotemporal or mechanical cues, and translating them into specific cellular outputs. These results provide an attractive model for how the Hippo cascade might sense and transduce cellular ‘neighborhood’ cues into activation of tissue-specific stem or progenitors cells. A further understanding of this process could allow the development of new therapies for various degenerative diseases and cancers. Here, we review current and emerging data linking Hippo signaling to progenitor cell function.     

Regulation of intestinal stem cell proliferation by human methyl-CpG-binding protein-2 in Drosophila

Recent studies have suggested the involvement of epigenetic factors such as methyl-CpG-binding protein-2 (MeCP2) in tumorigenesis. In addition, cancer may represent a stem cell-based disease, suggesting that understanding of stem cell regulation could provide valuable insights into the mechanisms of tumorigenesis. However, the function of epigenetic factors in stem cell regulation in adult tissues remains poorly understood. In the present study, we investigated the role of human MeCP2 (hMeCP2), a bridge factor linked to DNA modification and histone modification, in stem cell proliferation using adult Drosophila midgut, which appears to be an excellent model system to study stem cell biology. Results show that enterocyte (EC)-specific expression of hMeCP2 in adult midgut using an exogenous GAL4/UAS expression system induced intestinal stem cell (ISC) proliferation marked by staining with anti-phospho-histone H3 antibody and BrdU incorporation assays. In addition, hMeCP2 expression in ECs activated extracellular stress-response kinase signals in ISCs. Furthermore, expression of hMeCP2 modulated the distribution of heterochromatin protein-1 in ECs. Our data suggests the hypothesis that the expression of hMeCP2 in differentiated ECs stimulates ISC proliferation, implying a role of MeCP2 as a stem cell regulator.     

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU⁺ and Ki67⁺ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.     

The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation

The inherited human disease tuberous sclerosis, characterized by hamartomatous tumors, results from mutations in either TSC1 or TSC2. We have characterized mutations in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions.     

Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal

Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.     

Regulation of stem cell maintenance and transit amplifying cell proliferation by tgf-beta signaling in Drosophila spermatogenesis

The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells [1]. Previous data have suggested that TGF-beta signaling pathway components punt and schnurri are required in the somatic support cells to restrict germ cell proliferation. Here, by contrast, we show that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-beta ligand, the BMP5/8 ortholog Glass Bottom Boat. We further demonstrate that TGF-beta signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state.     

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.     

JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.