Alla Amcheslavsky

Tissue damage-induced intestinal stem cell division in Drosophila

Stem cell division is essential for tissue integrity during growth, aging, and pathogenic assaults. Adult gastrointestinal tract encounters numerous stimulations, and impaired tissue regeneration may lead to inflammatory diseases and cancer. Intestinal stem cells in adult Drosophila have recently been identified and shown to replenish the various cell types within the midgut. However, it is not known whether these intestinal stem cells can respond to environmental challenges. By feeding dextran sulfate sodium and bleomycin to flies and by expressing apoptotic proteins, we show that Drosophila intestinal stem cells can increase the rate of division in response to tissue damage. Moreover, if tissue damage results in epithelial cell loss, the newly formed enteroblasts can differentiate into mature epithelial cells. By using this newly established system of intestinal stem cell proliferation and tissue regeneration, we find that the insulin receptor signaling pathway is required for intestinal stem cell division.

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells.

Excessive cell growth in Drosophila intestinal stem cells lacking TSC blocks further cell division.

The Conserved Misshapen-Warts-Yorkie Pathway Acts in Enteroblasts to Regulate Intestinal Stem Cells in Drosophila

Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here, we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.

Enteroendocrine Cells Support Intestinal Stem-Cell-Mediated Homeostasis in Drosophila

Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. We show here that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth.

Interleukin (IL)-12 mediates the anti-osteoclastogenic activity of CpG-oligodeoxynucleotides

Bacterial DNA activates the innate immune system via interactions with Toll‐like receptor 9 (TLR9). This receptor recognizes CpG‐oligodeoxynucleotides (CpG‐ODNs) mimicking the CpG dinucleotides in certain sequence contexts characterizing this DNA. Most studies have shown increased osteoclast differentiation by TLR ligands. We found that activation of TLRs (specifically TLR4 and TLR9) in early osteoclast precursors results in inhibition of receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation. Our objective is to identify the mechanism leading to this inhibitory effect of a TLR ligand. Since both RANKL–RANK and CpG–ODN–TLR9 interactions result in NF‐κB activation, p38 and ERK phosphorylation, and TNF‐α synthesis (all implicated in osteoclastogenesis), we hypothesized that CpG‐ODN (but not RANKL) in addition induces the synthesis of an anti‐osteoclastogenic factor. Control osteoclast precursors, and cells treated with RANKL, CpG‐ODN, or their combination were studied using DNA arrays (GEArray Q Series Mouse NF‐κB Signaling Pathway Gene Array, MM‐016, SuperArray). We found a marked increase in the mRNA levels of the osteoclastogenesis inhibitor interleukin‐12 (IL‐12) in osteoclast precursors treated with CpG‐ODN and CpG‐ODN + RANKL. Northern and Western analyses, together with ELISA, confirmed the DNA array studies. In correlation with these findings, IL‐12 inhibited RANKL‐induced osteoclast differentiation and specific anti‐IL‐12‐antibodies inhibited the anti‐osteoclastogenic effect of CpG‐ODN. In conclusion, activation of TLR9 by its ligand, CpG‐ODN, results in synthesis and release of IL‐12 opposing RANKL‐induced osteoclast differentiation. J. Cell. Physiol. 207: 244–250, 2006.

Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1

The osteoclast is a highly polarized multinucleated cell that resorbs bone. Using high resolution immunofluorescence microscopy, we demonstrated that all nuclei of an osteoclast are transcriptionally active. Each nucleus within the osteoclast contains punctately organized microenvironments where regulatory complexes that support transcriptional and post‐transcriptional control reside. Functional equivalency of osteoclast nuclei is reflected by similar representation of regulatory proteins that support ribosomal RNA synthesis (nucleolin), mRNA transcription (RNA polymerase II, bromouridine triphosphate), processing of gene transcripts (SC35), signal transduction (NF‐κB), and phenotypic gene expression (Runx1). Our results establish that gene regulatory machinery is architecturally associated and compartmentalized within intranuclear microenvironments of the multiple nuclei of osteoclasts to support physiologically responsive modifications in cellular structural and functional properties.

Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.

CpG‐ODNs modulate osteoclast differentiation through Toll‐like receptor 9 (TLR9). Using TLR9‐deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG‐ODN. Osteoclastic TLR9 is more important for this activity.

Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.

CpG‐ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c‐fos, an essential molecule for osteoclast differentiation. Therefore, CpG‐ODN blocks osteoclast differentiation.

Be a Good Neighbor: Organ-to-Organ Communication during the Innate Immune Response

Local infection in the Drosophila larval intestine elicits a systemic immune reaction in fat bodies. In this issue, Wu and colleagues (2012) show that this is a reactive oxygen species-dependent communication.

Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila.

Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of EdU incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression of Charlatan blocked differentiation reversibly, but loss of Charlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation.