Drosophila twin spot clones reveal cell division dynamics in regenerating imaginal discs

Cell proliferation is required for tissue regeneration, yet the dynamics of proliferation during regeneration are not well understood. Here we investigated the proliferation of eye and leg regeneration in fragments of Drosophila imaginal discs. Using twin spot clones, we followed the proliferation and fates of sister cells arising from the same mother cell in the regeneration blastema. We show that the mother cell gives rise to two sisters that participate equally in regeneration. However, when cells switch disc identity and transdetermine to another fate, they fail to turn off the cell cycle and continue dividing long after regeneration is complete. We further demonstrate that the regeneration blastema moves as a sweep of proliferation, in which cells are displaced. Our results suggest that regenerating cells stop dividing once the missing parts are formed, but if they undergo a switch in cell fate, the proliferation clock is reset.     

Transdetermination in Drosophila imaginal discs: a model for understanding pluripotency and selector gene maintenance

Drosophila imaginal disc cells have the ability to undergo transdetermination, a process whereby determined disc cells change fate to that of another disc identity. For example, leg disc cells can transdetermine to develop as wing cells. Such events can occur after mechanical disc fragmentation and subsequent regeneration. A subset of transdetermination events can be induced in situ by misexpression of the signaling gene wingless. Both fragmentation and wingless induce transdetermination by altering the expression of selector genes, which drive disc-specific developmental programs. An important future goal is to address how signaling pathways interact with chromatin structure to regulate and maintain the proper expression of selector genes.     

Transdetermination: Drosophila imaginal disc cells exhibit stem cell-like potency

Drosophila imaginal discs, the primordia of the adult fly appendages, are an excellent system for studying developmental plasticity. Cells in the imaginal discs are determined for their disc-specific fate (wingness, legness) during embryogenesis. Disc cells maintain their determination during larval development, a time of extensive growth and proliferation. Only when prompted to regenerate do disc cells exhibit lability in their determined identity. Regeneration in the disc is mediated by a localized region of cell division, known as the regeneration blastema. Most regenerating disc cells strictly adhere to their disc-specific identity; some cells however, switch fate in a phenomenon known as transdetermination. Similar regeneration and transdetermination events can be induced in situ by misexpression of the signaling molecule wingless. Recent studies indicate that the plasticity of disc cells during regeneration is associated with high morphogen activity and the reorganization of chromatin structure. Here we provide both a historical perspective of imaginal disc transdetermination, as well as discuss recent findings on how imaginal disc cells acquire developmental plasticity and multipotency. We also highlight how an understanding of imaginal disc transdetermination can enhance an understanding of developmental potency exhibited by stem cells.     

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.     

Localization of cells capable of transdetermination in a specific region of the male foreleg disk ofDrosophila

Summary In the male foreleg disk ofDrosophila melanogaster the cells capable of transdetermination are clustered in a specific region within the upper half of the disk. Cells outside this region cannot transdetermine under any of the experimental conditions thus far applied.Transdetermination occurs when cells capable of transdetermination are stimulated to a certain extent of additional proliferation. This can be achieved either by exposing these cells at a wound surface of an intact fragment, or by dissociation.     

Regeneration and transdetermination: The role of wingless and its regulation

Imaginal discs of Drosophila have the remarkable ability to regenerate. After fragmentation wound healing occurs, ectopic wg is induced and a blastema is formed. In some, but not all fragments, the blastema will replace missing structures and a few cells can become more plastic and transdetermine to structures of other discs. A series of systematic cuts through the first leg disc revealed that a cut must transect the dorsal-proximal disc area and that the fragment must also include wg-competent cells. Fragments that fail to both transdetermine and regenerate missing structures will do both when provided with exogenous Wg, demonstrating the necessity of Wg in regenerative processes. In intact leg discs ubiquitously expressed low levels of Wg also leads to blastema formation, regeneration and transdetermination. Two days after exogenous wg induction the endogenous gene is activated, leading to elevated levels of Wg in the dorsal aspect of the leg disc. We identified a wg enhancer that regulates ectopic wg expression. Deletion of this enhancer increases transdetermination, but lowers the amount of ectopic Wg. We speculate that this lessens repression of dpp dorsally, and thus creates a permissive condition under which the balance of ectopic Wg and Dpp is favorable for transdetermination.     

Genetic analysis of transdetermination in Drosophila. I. The effects of varying growth parameters using a temperature-sensitive mutation

The heat-sensitive mutation of Drosophila melanogaster l(3)c4(3)hs1, causes mutant larvae raised at a restrictive temperature to have abnormally large wing discs. The large size of these discs is a disc-autonomous property and results from an increase in the number rather than the size of wing disc cells. We have used wing discs from this mutant to further investigate properties of transdetermination which had previously been investigated with nonmutant discs. Transdetermination can occur in nonmutant discs when the proliferative phase of imaginal disc development is extended by wounding discs and culturing them in vivo. The results indicate that additional proliferation in the absence of wounding does not lead to transdetermination. There is a correlation between the extent of growth of a cultured disc and the probability that it will undergo transdetermination. The results suggest that this correlation does not depend on a differential rate of cell division. Finally, the results indicate that the cells which give rise to transdetermination are at an equivalent developmental stage no later than that characteristic of eye-antenna disc cells before the third larval instar.     

Three genes control the timing, the site and the size of blastema formation in Drosophila

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless (wg/wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration (rgn), augmenter of liver regeneration (alr) and Matrix metalloproteinase-1 (Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.     

Divided loyalties: transdetermination and the genetics of tissue regeneration

Most tissues contain cells capable of the self-renewal and differentiation necessary to maintain tissue and organ integrity. These somatic stem cells are generally thought to have limited developmental potential. The mechanisms that restrict cell fate decisions in somatic stem cells are only now being understood. This understanding will be important in the clinical exploitation of adult stem cells in tissue repair and replacement. Experiments performed over fifty years ago in Drosophila showed that developmental restriction could be relaxed in the proliferating larval cells that are destined to form the adult fly integument. This phenomenon, called transdetermination, can serve as a model for mechanisms of stem-cell commitment. A recent publication (1) sheds new light on the mechanism of transdetermination by demonstrating that loss of homeotic gene silencing leads to increased frequency of transdetermination. In addition, the authors link a specific signaling pathway induced by tissue regeneration to the relaxation of homeotic gene silencing. The data identify key mechanisms that control developmental homeostasis and cell fate restriction that could be manipulated to make somatic stem-cell engineering possible.     

Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state

Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.     

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. We show that the homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut was expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression was controlled by the Notch pathway and was essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells entered the endocycle and differentiated prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut caused defects in the mitotic cycle/endocycle switch. These cells continued to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promoted Cyclin A expression by negatively regulating Fizzy-related. Our data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells.     

Genetic modifiers of ectopic eye formation on wings of Drosophila melanogaster

The ectopic expression of the master ey gene by the GAL4-UAS system can induce ectopic eye formation in different organs. The formation of ectopic eyes takes place in certain regions of imaginal discs, which partially overlap with the regions responsible for the transdetermination of differentiated cells (essentially meaning the alteration of the cell fate). In this way, ectopic eye induction could be considered as a model for cellular plasticity studies. In the present work, we performed a search for transgenes, the ectopic coexpression of which with the master ey gene induced morphologic changes in the ectopic eyes on the wing compared to the sole ey expression. Most of the transgenes found to affect the size of ectopic eyes belonged to the class of vesicular trafficking genes capable of affecting different signaling pathways. The ectopic expression of the revealed transgenes in the wing and eye discs altered the morphology of both normal wings and normal eyes. We argue that the effect of these genes may be that they change the size of the region responsible for cell fate transdetermination.     

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.     

Temporal regulation of cerebellar EGL migration through a switch in cellular responsiveness to the meninges

We have studied the temporal and spatial control of cell migration from the external germinal layer (EGL) in the mammalian cerebellum as a model for cortical migration. Our results have demonstrated that embryonic EGL cells do not migrate into internal layers because they respond to a diffusible attractant in the meninges, the nonneural tissues covering the nervous system, and to a repellent in the neuroepithelium. Two developmental changes are important for postnatal EGL migration: the disappearance of the repellent in the inner layers and a switch in cellular responsiveness of EGL cells so that the postnatal EGL cells respond to the repellent, but not the attractant in the meninges. Besides revealing the signaling role of meninges in cortical development, our study suggests that an active mechanism is required to prevent cell migration, and that mechanisms of cell migration should be studied even in the absence of apparent changes in cell positions. We propose a model for the developmental control of neuronal migration in the cerebellar cortex.     

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.     

Paneth cells in intestinal homeostasis and tissue injury

Adult stem cell niches are often co-inhabited by cycling and quiescent stem cells. In the intestine, lineage tracing has identified Lgr5(+) cells as frequently cycling stem cells, whereas Bmi1(+), mTert(+), Hopx(+) and Lrig1(+) cells appear to be more quiescent. Here, we have applied a non-mutagenic and cell cycle independent approach to isolate and characterize small intestinal label-retaining cells (LRCs) persisting in the lower third of the crypt of Lieberkühn for up to 100 days. LRCs do not express markers of proliferation and of enterocyte, goblet or enteroendocrine differentiation, but are positive for Paneth cell markers. While during homeostasis, LR/Paneth cells appear to play a supportive role for Lgr5(+) stem cells as previously shown, upon tissue injury they switch to a proliferating state and in the process activate Bmi1 expression while silencing Paneth-specific genes. Hence, they are likely to contribute to the regenerative process following tissue insults such as chronic inflammation.