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.     

Spatio-temporal pattern of programmed cell death in the developing Drosophila optic lobe

A large number of cells die via programmed cell death during the normal development of the Drosophila optic lobe. In this study, we report the precise spatial and temporal pattern of cell death in this organ. Cell death in the developing optic lobe occurs in two distinct phases. The first phase extends from the start of metamorphosis to the mid-pupal stage. During this phase, a large number of cells die in the optic lobe as a whole, with a peak of cell death at an early pupal stage in the lamina and medulla cortices and the region of the T2/T3/C neurons, and a smaller number of dead cells observed in the lobula plate cortex. The second phase extends from the mid-pupal stage to eclosion. Throughout this period, a small number of dying cells can be observed, with a small peak at a late pupal stage. Most of the dying cells are neurons. During the first phase, dying cells are distributed in specific patterns in cortices. The lamina cortex contains two distinct clusters of dying cells; the medulla cortex, four clusters; the lobula plate cortex, one cluster; and the region of the T2/T3/C neurons, one cluster. Many of the clusters maintain their distinct positions in the optic lobe but others extend the region they cover during development. The presence of distinct clusters of dying cells at different phases suggests that distinct mechanisms control cell death during different stages of optic lobe development in Drosophila.     

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.     

Benzodiazepine receptor sites in the chick optic lobe: Development and pharmacological characterization

To investigate the, interaction between -aminobutyric acid (GABA) and benzodiazepine (BZD) receptor sites during development, the time-course of appearance of flunitrazepam (FNZ) binding sites and their pharmacological characterization were studied in developing chick optic lobe. At the earliest stage examined, embryonic day (Ed) 12, the receptor density was 30.9 % (0.05±0.01 pmol/mg protein) of that found in the chick optic lobes of adult chicks. The adult value was achieved on Ed 16 (0.16±0.01 pmol/mg protein). After this stage there was a sharp and transient increase in specific [³H]FNZ binding of about two-fold reaching a maximal value between hatching and the postnatal day (pnd) 2 (0.33±0.01 pmol/mg protein). Scatchard analysis at different stages of development revealed the presence of a single population of specific FNZ binding sites. The increase in [³H]FNZ binding during development was due to a large number of binding sites while their affinity remained unchanged. Competition experiments in the chick optic lobe revealed that the order of potency for displacement of specific [³H]FNZ binding paralleled the pharmacological potency of the BZDs tested. The IC₅₀ s for clonazepam, flunitrazepam, Ro 15-1788 and chlordiazepoxide were 3.02, 4.30, 0.32, and 4778.64 nM respectively. Ro 5-4864, a potent inhibitor of BZD binding to peripheral tissues, had no effect on specific [³H]FNZ binding indicating that only central BZD binding sites are present in the chick optic lobe. The peak of maximal expression of BZD receptor sites precedes in 5–6 days the peak of GABA receptor sites indicating a precocious development of BZD receptor sites. The different appearance of both peaks may represent important events during development probably related to synaptogenesis.     

Downregulation of Notch mediates the seamless transition of individual Drosophila neuroepithelial progenitors into optic medullar neuroblasts during prolonged G1

The first step in the development of the Drosophila optic medullar primordia is the expansion of symmetrically dividing neuroepithelial cells (NEs); this step is then followed by the appearance of asymmetrically dividing neuroblasts (NBs). However, the mechanisms responsible for the change from NEs to NBs remain unclear. Here, we performed detailed analyses demonstrating that individual NEs are converted into NBs. We also showed that this transition occurs during an elongated G1 phase. During this G1 phase, the morphological features and gene expressions of each columnar NE changed dynamically. Once the NE-to-NB transition was completed, the former NE changed its cell-cycling behavior, commencing asymmetric division. We also found that Notch signaling pathway was activated just before the transition and was rapidly downregulated. Furthermore, the clonal loss of the Notch wild copy in the NE region near the medial edge caused the ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, these findings indicate that the activation of Notch signaling during a finite window coordinates the proper timing of the NE-to-NB transition.     

Neurosecretory cells in the optic lobe of two aquatic coleopteran species,Dineutes indicus aube and Cybister rugulosus redt

In each optic lobe and optic peduncle of two aquatic beetles viz. Dineutes indicus and Cybister rugulosus the neurosecretory cells are observed with the help of various histochemical techniques. These cells are arranged to form a discrete group. A group in the optic lobe of both species contains about 25 to 30 neurosecretory cells. On the basis of staining properties the neurosecretory cells are classified into A and B types. These cells stain with chrome haematoxylin-phloxine and paraldehyde fuchsin, but do not stain with azan. Histochemically, the neurosecretory material is positive for proteins and shows a negative reaction for 1,2-glycols. The cells show variations in RNA contents in correlation with the state of secretory activity. Axons of the neurosecretory cell group of the optic lobe are observed directed to the optic peduncle. The axonal tract from neurosecretory cells in the optic peduncle runs towards the lateral margin of the brain.     

Acceleration of brain tubulin formation in the optic lobe of the chick embryo by light stimulation

The effect of light exposure on the protein patterns of optic lobe and forebrain of the chick embryo was analysed by a high-resolution micro-two-dimensional polyacryamide gel electrophoresis and computerized quantitation. Experiments were done on three groups of eggs: control group was incubated in the dark; simultaneously, in the same incubator, one group of eggs was illuminated by constant light, another by intermittent light (3 sec interval) from day 10 to day 16 of incubation. In embryos exposed to intermittent light the relative amount of tubulins was significantly increased in the optic lobe. In the frontal lobe no effect of light exposure on the concentration of tubulins was seen. The rise of tubulins in the optic lobe was only caused by intermittant light. Continuous illumination of the eggs for the same period under otherwise identical incubation conditions was ineffective.Abbreviations used BSA Bovine serum albumin - CBB Coomassie Brilliant Blue G-250 - 2D-PAGE Two dimensional polyacrylamide gel electrophoresis - IEF Isoelectric focusing - M_r Molecular weight - pI Isoelectric point - SDS Sodium dodecyl sulfate - TEMED N,N,N,N-Tetramethylethyenediamine     

Autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway

Autophagy plays important roles in self-renewal and differentiation of stem cells. Hepatic progenitor cells (HPCs) are thought to have the ability of self-renewal as well as possess a bipotential capacity, which allows them to differentiate into both hepatocytes and bile ductular cells. However, how autophagy contributes to self-renewal and differentiation of hepatic progenitor cells is not well understood. In this study, we use a well-established rat hepatic progenitor cell lines called WB-F344, which is treated with 3.75 mM sodium butyrate (SB) to promote the differentiation of WB-F344 along the biliary phenotype. We found that autophagy was decreased in the early stage of biliary differentiation, and maintained a low level at the late stage. Activation of autophagy by rapamycin or starvation suppressed the biliary differentiation of WB-F344. Further study reported that autophagy inhibited Notch1 signaling pathway, which contributed to biliary differentiation and morphogenesis. In conclusions, autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.     

The optic lobe of Drosophila melanogaster

Summary We present a quantitative evaluation of Golgiimpregnated columnar neurons in the optic lobe of wildtype Drosophila melanogaster. This analysis reveals the overall connectivity pattern between the 10 neuropil layers of the medulla and demonstrates the existence of at least three major visual pathways. Pathway 1 connects medulla layer M10 to the lobula plate. Input layers of this pathway are M1 and M5. Pathway 2 connects M9 to shallow layers of the lobula, which in turn are tightly linked to the lobula plate. This pathway gets major input via M2. Pathways 1 and 2 receive input from retinula cells R1-6, either via the lamina monopolar cell L1 (terminating in M1 and M5) or via L2 and T1 (terminating in M2). Neurons of these pathways typically have small dendritic fields. We discuss evidence that pathways 1 and 2 may play a major role in motion detection. Pathway 3 connects M8 to deep layers of the lobula. In M8 information converges that is derived either from M3 (pathway 3a) or from M4 and M6 (pathway 3b), layers that get their major input from L3 and R8 or L4 and R7, respectively. Some neurons of pathway 3 have large dendritic fields. We suggest that they may be involved in the computation of form and colour. Possible analogies to the organization of pathways in the visual system of vertebrates are discussed.During the final editing of this work our friend A.P.M. Dittrich was tragically killed in an accident. Without him this and the previous work would never have been completed     

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell species differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and we refer to its signal array as the "proneural wave," as it is the marker of the EGFR/Ras signaling front. In this study, we show that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. We also show that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system.     

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

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.     

Cooperation of JAK/STAT and Notch signaling in the Drosophila foregut

Temporal and spatial regulation of morphogenesis is pivotal to the formation of organs from simple epithelial tubes. In a genetic screen for novel genes controlling cell movement during posterior foregut development, we have identified and molecularly characterized two alleles of the domeless gene which encodes the Drosophila Janus kinase (JAK)/STAT receptor. We demonstrate that mutants for domeless or any other known component of the canonical JAK/STAT signaling pathway display a failure of coordinated cell movement during the development of the proventriculus, a multiply folded organ which is formed by stereotyped cell rearrangements in the posterior foregut. Whereas the JAK/STAT receptor is expressed in all proventricular precursor cells, expression of upd encoding its ligand and of STAT92E, the signal transducer of the pathway, is locally restricted to cells that invaginate during proventriculus development. We demonstrate by analyzing gene expression mediated by a model Notch response element and by studying the expression of the Notch target gene short stop, which encodes a cytoskeletal crosslinker protein, that JAK/STAT signaling is required for the activation of Notch-dependent gene expression in the foregut. Our results provide strong evidence that JAK/STAT and Notch signaling cooperate in the regulation of target genes that control epithelial morphogenesis in the foregut.     

The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila

Notch (N) is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and was proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. Here, we report the first null mutant allele of Drosophila dx. We found that dx was involved only in the subsets of N signaling, but was not essential for it in any developmental context. A strong genetic interaction between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. Our epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). We also uncovered a novel dx activity that suppressed N signaling downstream of N.     

Notch signaling plays a key role in cardiac cell differentiation

Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Cardiac conduction cells are specialized cells responsible for orchestrating the rhythmic contractions of the heart. Here, we show that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker, HNK-1, at early stages. Misexpression of constitutively active Notch1 (NIC) in early heart tubes in chick exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in expression of cardiac muscle markers, but an increase in expression of conduction cell markers, HNK-1, and SNAP-25. However, the expression of the conduction marker connexin 40 was inhibited. Loss-of-function study, using a dominant-negative form of Suppressor-of-Hairless, further supports that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.     

Neural pathways involved in mutual interactions between optic lobe circadian pacemakers in the cricketGryllus bimaculatus

The circadian locomotor rhythm of the cricketGryllus bimaculatus is primarily generated by a pair of optic lobe circadian pacemakers. The two pacemakers mutually interact to keep a stable temporal structure in the locomotor activity. The interaction has two principal effects on the activity rhythm, i.e., phase-dependent modulation of the freerunning period and phase-dependent suppression of activity driven by the partner pacemaker. Both effects were mediated by neural pathways, since they were immediately abolished after the optic stalk connecting the optic medulla to the lobula was unilaterally severed. The neural pathways were examined by recording locomotor activity, under a 13 h light to 13 h dark cycle, after the optic nerves were unilaterally severed and the contralateral optic stalk was partially destroyed near the lobula. When the dorsal half of the optic stalk was severed, locomotor rhythm mostly split into two components: one was readily entrained to the given light-dark cycle and the other freeran with a marked fluctuation in freerunning period, where the period of the freerunning component was lengthened or shortened when the onset of the entrained component occurred during its subjective night or day, respectively. The phase-dependent modulation of activity was also observed in both components. However, severance of the ventral half of the optic stalk resulted in appearance only of the freerunning component; neither the phase-dependent modulation of its freerunning period nor the change in activity level was observed. These results suggest that neurons driving the mutual interaction and the overt activity rhythm run in the ventral half of the proximal optic stalk that includes axons of large medulla neurons projecting to the cerebral lobe and the contralateral medulla.Abbreviations LD light dark cycle - freerunning period     

Notch signaling regulates growth and differentiation in the mammalian lens

The Notch signal transduction pathway regulates the decision to proliferate versus differentiate. Although there are a myriad of mouse models for the Notch pathway, surprisingly little is known about how these genes regulate early eye development, particularly in the anterior lens. We employed both gain-of-function and loss-of-function approaches to determine the role of Notch signaling in lens development. Here we analyzed mice containing conditional deletion of the Notch effector Rbpj or overexpression of the activated Notch1 intracellular domain during lens formation. We demonstrate distinct functions for Notch signaling in progenitor cell growth, fiber cell differentiation and maintenance of the transition zone. In particular, Notch signaling controls the timing of primary fiber cell differentiation and is essential for secondary fiber cell differentiation. Either gain or loss of Notch signaling leads to formation of a dysgenic lens, which in loss-of-function mice undergoes a profound postnatal degeneration. Our data suggest both Cyclin D1 and Cyclin D2, and the p27^Kip1 cyclin-dependent kinase inhibitor act downstream of Notch signaling, and define multiple critical functions for this pathway during lens development.     

RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-Jκ (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.