Transiently Undead Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut

We reveal surprising similarities between homeostatic cell turnover in adult Drosophila midguts and undead apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. Here, we show that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. We propose that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently undead cells may be relevant for other stem cell models in flies and mammals

Tumor-promoting function of apoptotic caspases by an amplification loop involving ROS, macrophages and JNK in Drosophila

Apoptosis and its molecular mediators, the caspases, have long been regarded as tumor suppressors and one hallmark of cancer is \u27Evading Apoptosis\u27. However, recent work has suggested that apoptotic caspases can also promote proliferation and tumor growth under certain conditions. How caspases promote proliferation and how cells are protected from the potentially harmful action of apoptotic caspases is largely unknown. Here, we show that although caspases are activated in a well-studied neoplastic tumor model in Drosophila, oncogenic mutations of the proto-oncogene Ras (Ras(V12)) maintain tumorous cells in an \u27undead\u27-like condition and transform caspases from tumor suppressors into tumor promotors. Instead of killing cells, caspases now promote the generation of intra- and extracellular reactive oxygen species (ROS). One function of the ROS is the recruitment and activation of macrophage-like immune cells which in turn signal back to tumorous epithelial cells to activate oncogenic JNK signaling. JNK further promotes and amplifies caspase activity, thereby constituting a feedback amplification loop. Interfering with the amplification loop strongly reduces the neoplastic behavior of these cells and significantly improves organismal survival. In conclusion, Ras(V12)-modified caspases initiate a feedback amplification loop involving tumorous epithelial cells and macrophage-like immune cells that is necessary for uncontrolled tumor growth and invasive behavior

Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

BACKGROUND: ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer. RESULTS: Here, we show that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development. CONCLUSION: In addition to a role of dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP

Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc(+) in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis

Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila

Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Additional file 3: Figure S3. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Loss of dAtg1 does not suppress apoptosis. (A–B’) Mosaic late third instar wing discs with hid-p35-expressing clones positively marked by GFP. Simultaneous expression of hid and p35 in clones induces strong cCasp3 labeling (A, A’, arrows). Similar cCasp3 labeling persists in dAtg1 mutant clones (B, B’, arrows). (C) Quantification of cCasp3 labeling intensity in hid-p35-expressing clones and hid-p35-expressing dAtg1 mutant clones (mean ± SE). No significant difference of cCasp3 labeling was observed. (D–D’) A representative late third instar GMR-hid eye disc with dAtg1 mutant clones negatively marked by GFP (highlighted by yellow dotted lines). The wave of apoptosis (arrow) induced by GMR-hid persists in dAtg1 mutant clones. (E, F) Representative adult eyes of the indicated genotypes. GMR-hid-induced eye ablation phenotype (E) is not altered by RNAi knockdown of dAtg1 (F). (TIF 6416 kb

Additional file 6: Figure S6. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

(A–C”) Autophagic flux reporter expression in ey > hid-p35 eye discs. Late third instar larval eye discs expressing the autophagic flux reporter GFP-mCherry-dAtg8a under control of the dAtg8 promoter [76]. The yellow dotted lines indicate the anterior portions of the eye discs which expresses ey-Gal4. Note the overgrowth of the anterior eye disc portion in ey > hid-p35 imaginal discs (B–B”). Expression of GFP and mCherry is low in the control ey > p35 discs (A–A”). In contrast, the numbers of GFP and mCherry positive particles are strongly increased in the overgrown ey-Gal4 expressing area of ey > hid-p35 discs (B–B”). Although the overgrowth of ey > hid-p35 eye discs is strongly suppressed by dAtg1 RNAi, the GFP and mCherry signals are not significantly reduced (C–C”). (TIF 7456 kb

Additional file 7: Figure S7. of Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation

Functional tests of the RNAi lines targeting dAtg1, dAg3, dAtg8a, and dAtg8b. (A–E) Starvation assay of fat bodies from third instar larvae. Formation of autophagosomes was visualized by mCherry-Atg8 (red in A–E; grey in A’–E’). Cells expressing RNAi constructs are labeled by GFP and outlined by yellow dotted lines. (A) Wildtype fat body displaying mCherry-Atg8 puncta both in clone cells and surrounding cells. (B–E) Cells expressing dAtg1, dAtg3, dAtg8a, and dAtg8b RNAi (GFP +) fail to form mCherry-Atg8 marked autophagosomes. The loss of mCherry-Atg8 signals by dAtg8a and dAtg8b RNAi in (D) and (E) also demonstrates that these RNAi lines target mCherry-Atg8 transcripts. (F–J) Adult eyes expressing eyeful and indicated RNAi transgenes. As previously reported [77], loss of autophagy strongly enhances the eyeful phenotype. The functionality of dAtg1, dAtg3, dAtg8a, and dAtg8b RNAi transgenes is confirmed by enhancement of the eyeful phenotype. (TIF 8420 kb