Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Neural stem cells called neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. Here, we show that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered ;proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. Our findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center.     

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.     

Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe

The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. Our findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions.     

Loss of Rab5 drives non-autonomous cell proliferation through TNF and Ras signaling in Drosophila

Deregulation of the endocytic machinery has been implicated in human cancers. However, the mechanism by which endocytic defects drive cancer development remains to be clarified. Here, we find through a genetic screen in Drosophila that loss of Rab5, a protein required for early endocytic trafficking, drives non-autonomous cell proliferation in imaginal epithelium. Our genetic data indicate that dysfunction of Rab5 leads to cell-autonomous accumulation of Eiger (a TNF homolog) and EGF receptor (EGFR), which causes activation of downstream JNK and Ras signaling, respectively. JNK signaling and its downstream component Cdc42 cooperate with Ras signaling to induce upregulation of a secreted growth factor Upd (an IL-6 homolog) through inactivation of the Hippo pathway. Such non-autonomous tissue growth triggered by Rab5 defect could contribute to epithelial homeostasis as well as cancer development within heterogeneous tumor microenvironment.     

Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development

The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.     

Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila

Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. We demonstrate that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition.     

Interplay between Hsp90 and TPR domain-containing proteins in steroidogenic signaling

EGFL7 drives the formation of neurons from neural stem cells. In the embryonic and adult brain this process is essential for neurogenesis and homeostasis of the nervous system. The function of adult neurogenesis is not fully understood but maybe it supports life-long learning and brain repair after injuries such as stroke. The transition of neural stem cells into mature neurons is tightly regulated. One of the essential signaling pathways governing this process is the Notch pathway, which controls metazoan development. In a recent publication, we identified a novel non-canonical Notch ligand, EGFL7, and described its impact on neural stem cells.¹ We explored the molecular mechanisms, which this molecule affects to regulate the self-renewal capacity of neural stem cells and to promote their differentiation into neurons. In this review, we discuss the implications of our findings for adult neurogenesis and illustrate the potential of EGFL7 to serve as an agent to increase neurogenesis and the self-renewal potential of the brain.     

The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. Here we show that the proneural proteins trigger, in addition, positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (EGFR) and the Ras/Raf pathway. These interactions, which we denominate 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the EGFR/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess EGFR signalling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the EGFR and Notch pathways act antagonistically in notum macrochaetae determination.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

Regulating the balance between symmetric and asymmetric stem cell division in the developing brain

Stem cells proliferate through symmetric division or self-renew through asymmetric division whilst generating differentiating cell types. The balance between symmetric and asymmetric division requires tight control to either expand a stem cell pool or to generate cell diversity. In the Drosophila optic lobe, symmetrically dividing neuroepithelial cells transform into asymmetrically dividing neuroblasts. The switch from neuroepithelial cells to neuroblasts is triggered by a proneural wave that sweeps across the neuroepithelium. Here we review recent findings showing that the orchestrated action of the Notch, EGFR, Fat-Hippo, and JAK/STAT signalling pathways controls the progression of the proneural wave and the sequential transition from symmetric to asymmetric division. The neuroepithelial to neuroblast transition in the optic lobe bears many similarities to the switch from neuroepithelial cell to radial glial cell in the developing mammalian cerebral cortex. The Notch signalling pathway has a similar role in the transition from proliferating to differentiating stem cell pools in the developing vertebrate retina and in the neural tube. Therefore, findings in the Drosophila optic lobe provide insights into the transitions between proliferative and differentiative division in the stem cell pools of higher organisms.     

BMP and Delta/Notch signaling control the development of amphioxus epidermal sensory neurons: insights into the evolution of the peripheral sensory system

The evolution of the nervous system has been a topic of great interest. To gain more insight into the evolution of the peripheral sensory system, we used the cephalochordate amphioxus. Amphioxus is a basal chordate that has a dorsal central nervous system (CNS) and a peripheral nervous system (PNS) comprising several types of epidermal sensory neurons (ESNs). Here, we show that a proneural basic helix-loop-helix gene (Ash) is co-expressed with the Delta ligand in ESN progenitor cells. Using pharmacological treatments, we demonstrate that Delta/Notch signaling is likely to be involved in the specification of amphioxus ESNs from their neighboring epidermal cells. We also show that BMP signaling functions upstream of Delta/Notch signaling to induce a ventral neurogenic domain. This patterning mechanism is highly similar to that of the peripheral sensory neurons in the protostome and vertebrate model animals, suggesting that they might share the same ancestry. Interestingly, when BMP signaling is globally elevated in amphioxus embryos, the distribution of ESNs expands to the entire epidermal ectoderm. These results suggest that by manipulating BMP signaling levels, a conserved neurogenesis circuit can be initiated at various locations in the epidermal ectoderm to generate peripheral sensory neurons in amphioxus embryos. We hypothesize that during chordate evolution, PNS progenitors might have been polarized to different positions in various chordate lineages owing to differential regulation of BMP signaling in the ectoderm.     

Fat cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo signaling pathway

BACKGROUND: The atypical Fat cadherin has long been known to control cell proliferation and organ size in Drosophila, but the mechanism by which Fat controls these processes has remained elusive. A newly emerging signaling pathway that controls organ size during development is the Salvador/Warts/Hippo pathway. RESULTS: Here we demonstrate that Fat limits organ size by modulating activity of the Salvador/Warts/Hippo pathway. ft interacts genetically with positive and negative regulators of this pathway, and tissue lacking fat closely phenocopies tissue deficient for genes that normally promote Salvador/Warts/Hippo pathway activity. Cells lacking fat grow and proliferate more quickly than their wild-type counterparts and exhibit delayed cell-cycle exit as a result of elevated expression of Cyclin E. fat mutant cells display partial insensitivity to normal developmental apoptosis cues and express increased levels of the anti-apoptotic DIAP1 protein. Collectively, these defects lead to increased organ size and organism lethality in fat mutant animals. Fat modulates Salvador/Warts/Hippo pathway activity by promoting abundance and localization of Expanded protein at the apical membrane of epithelial tissues. CONCLUSIONS: Fat restricts organ size during Drosophila development via the Salvador/Warts/Hippo pathway. These studies aid our understanding of developmental organ size control and have implications for human hyperproliferative disorders, such as cancers.     

Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras‐mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP‐SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras^V12 depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.