A mechanism for morphogen-controlled domain growth

Many developmental systems are organised via the action of graded distributions of morphogens. In the Drosophila wing disc, for example, recent experimental evidence has shown that graded expression of the morphogen Dpp controls cell proliferation and hence disc growth. Our goal is to explore a simple model for regulation of wing growth via the Dpp gradient: we use a system of reaction-diffusion equations to model the dynamics of Dpp and its receptor Tkv, with advection arising as a result of the flow generated by cell proliferation. We analyse the model both numerically and analytically, showing that uniform domain growth across the disc produces an exponentially growing wing disc.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

The Transcription Factor Optomotor-Blind Antagonizes Drosophila Haltere Growth by Repressing Decapentaplegic and Hedgehog Targets

In Drosophila, decapentaplegic, which codes for a secreted signaling molecule, is activated by the Hedgehog signaling pathway at the anteroposterior compartment border of the two dorsal primordia; the wing and the haltere imaginal discs. In the wing disc, Decapentaplegic and Hedgehog signaling targets are implicated in cell proliferation and cell survival. However, most of their known targets in the wing disc are not expressed in the haltere disc due to their repression by the Hox gene Ultrabithorax. The T-box gene optomotor-blind escapes this repression in the haltere disc, and therefore is expressed in both the haltere and wing discs. Optomotor-blind is a major player during wing development and its function has been intensely investigated in this tissue, however, its role in haltere development has not been reported so far. Here we show that Optomotor-blind function in the haltere disc differs from that in the wing disc. Unlike its role in the wing, Optomotor-blind does not prevent apoptosis in the haltere but rather limits growth by repressing several Decapentaplegic and Hedgehog targets involved both in wing proliferation and in modulating the spread of morphogens similar to Ultrabithorax function but without disturbing Ultrabithorax expression.     

A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. Here we address this possibility in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, Tkv(Q253D), causes overgrowth. We extend these findings with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing Tkv(Q253D) accelerated wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slowed wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by Tkv(Q253D) was blocked by the cell cycle inhibitor RBF, and required normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for Tkv(Q253D)-induced growth. The growth response to altering Dpp signaling varied regionally and temporally in the wing disc, indicating that other patterned factors modify the response.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Dissecting the mechanisms of Notch induced hyperplasia

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch‐CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non‐autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed‐forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross‐regulatory relationships are important in coordinating the pattern of hyperplasia.     

Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.     

Truncated products of the vestigial proliferation gene induce apoptosis.

The vestigial (vg) gene in D. melanogaster, whose mutant phenotype is characterized by wing atrophy, encodes a novel nuclear protein involved in cell proliferation. The original vg mutant (vgBG) displays massive apoptosis in the wing imaginal disc. Here we tested the hypothesis that the vg mutant phenotype could be due: (i) to lack of cell proliferation in null mutants due to the absence of the Vg product and, (ii) to apoptosis in vgBG and other mutants due to the presence of a major Vg truncated product. In agreement with our hypothesis no cell death was observed in null vg mutants, and the anticell death baculovirus P35 product is unable to rescue the mutant phenotype caused by absence of the Vg product. In addition, expression of the antiproliferative gene dacapo, the homolog of p21, induces a mutant wing phenotype without inducing cell death. In contrast the wing phenotype of the original vg mutant could be reproduced by the ectopic expression of the reaper cell death gene when expressed by vg regulatory sequences. In agreement with the hypothesis, the classic vg mutant spontaneously displays an increase in reaper expression in the wing disc and its phenotype can be partially rescued by the P35 product. Finally, we showed that ectopic expression of a truncated Vg product is able on its own to induce ectopic cell death and reaper expression. Our results shed new light on the function of the vg gene, in particular, they suggest that the normal and truncated products affect vg target genes in different ways.     

Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc

When a fragment of a Drosophila imaginal disc is cultured in growth permissive conditions, it either regenerates the missing structures or duplicates the pattern present in the fragment. This kind of pattern regulation is known to be epimorphic, i.e. the new pattern is generated by proliferation in a specialized tissue called the blastema. Pattern regulation is accompanied by the healing of the cut surfaces restoring the continuous epithelia. Wound healing has been considered to be the inductive signal to commence regenerative cell divisions. Although the general outlines of the proliferation dynamics in a regenerating imaginal disc blastema have been well studied, little is known about the mechanisms driving cells into the regenerative cell cycles. In this study, we have investigated the role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila wing imaginal disc. By utilizing in vivo and in vitro culturing of incised and fragmented discs, we have been able to visualize the dynamics in cellular architecture and gene expression involved in the healing and regeneration process. Our results directly show that homotypic wound healing is not a prerequisite for regenerative cell divisions. We also show that JNK signaling participates in imaginal disc wound healing and is regulated by the physical dynamics of the process, as well as in recruiting cells into the regenerative cell cycles. A model describing the determination of blastema size is discussed.     

Wingless and Notch signaling provide cell survival cues and control cell proliferation during wing development

Tissue growth during animal development depends on the coordination of cell proliferation and cell death. The EGF-receptor/MAPK, Hedgehog, Dpp, Wingless (Wg) and Notch signaling pathways have been implicated in growth control in the developing Drosophila wing. In this report, we examine the effects of Notch and Wg on growth in terms of cell proliferation and cell survival. Reduction of Wg signaling impaired compartment and clonal growth, and increased cell death. Inhibition of apoptosis in cells deficient for Wg signaling only partially rescued the clone growth defect, suggesting that Wg is also required to promote cell proliferation. This is supported by the finding that ectopic expression of Wg caused over-proliferation of cells in the proximal wing. Localized activation of Notch had non-autonomous effects on cell proliferation. However, only part of this effect was attributable to Notch-dependent induction of Wg, suggesting that other Notch-inducible signaling molecules contribute to the control of cell proliferation in the wing.     

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.     

The missing link: implementation of morphogenetic growth control on the cellular and molecular level

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.     

Compensatory proliferation induced by cell death in the Drosophila wing disc requires activity of the apical cell death caspase Dronc in a nonapoptotic role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%-60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc-in the absence of cell death-results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well.     

Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis

Flow cytometric analyses of imaginal disc and brain nuclei of Drosophila melanogaster have been made throughout the third larval instar. In wing, haltere, and leg discs the proportion of cells in the G₂M phase of the cell cycle (tetraploid cells) increases with larval age. In contrast, in the eye disc and in brain the proportion of tetraploid cells, already low at the outset of the instar, declines further. Measurement of growth rates for disc and brain tissue during the same developmental period was carried out by the cell counting procedure of Martin (1982). Our results are consistent with the conclusion that imaginal discs grow exponentially with an apparent doubling time of 5–10 hr from the resumption of cell division (in the first or second larval instar) until about 95 hr, when the apparent doubling time increases. Cell numbers increase until at least 5 hr after formation of white prepupae (122 hr), but during the preceding 10 hr the rate of increase is low. Thus, for wing and leg discs, but not for the eye disc and brain, the declining growth rate is associated with an increase in the proportions of tetraploid cells. In conjunction with cell counts and flow cytometry, fluorometric determination of disc DNA content at 112 hr indicated that the diploid DNA content of imaginal disc nuclei is 0.45 pg.     

My what big eyes you have: how the Drosophila retina grows

The compound eye of the fruit fly, Drosophila melanogaster, has for decades been used extensively to study a number of critical developmental processes including tissue development, pattern formation, cell fate specification, and planar cell polarity. To a lesser degree it has been used to examine the cell cycle and tissue proliferation. Discovering the mechanisms that balance tissue growth and cell death in developing epithelia has traditionally been the realm of those using the wing disc. However, over the last decade a series of observations has demonstrated that the eye is a suitable and maybe even preferable tissue for studying tissue growth. This review will focus on how growth of the retina is controlled by the genes and pathways that govern the specification of tissue fate, the division of the epithelium into dorsal-ventral compartments, the initiation, and progression of the morphogenetic furrow and the second mitotic wave.