The Interactive Fly

Zygotically transcribed genes

Proneural and Neurogenic Genes

What is the Notch pathway?
Developmental biology of the Notch pathway
The achaete-scute complex
Embryonic central nervous system lineages
Aging neural progenitors lose competence to respond to mitogenic Notch signaling
BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development
Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis
The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain
Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam
Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation
Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling
The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking
Drosophila chromatin assembly factor 1 p105 and p180 subunits are required for follicle cell proliferation via inhibiting Notch signaling>
An agent-based model of the Notch signaling pathway elucidates three levels of complexity in the determination of developmental patterning
Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23

Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity
Activation of Arp2/3 by WASp is essential for the endocytosis of Delta only during cytokinesis in Drosophila

Neurogenic genes functioning in the Notch pathway

Proneural genes of the achaete-scute complex

Additional proneural genes

What is the Notch pathway?

To understand the Notch pathway one has first to know the function of receptors and ligands. A receptor is a protein molecule located partially outside the cell and partially inside. The receptor interacts with outside signals and transmits them to the inside. Ligands are extracellular proteins or other molecules that interact with a receptor to cause it to transmit a signal to the inside of the cell. Notch is a receptor that receives signals from its ligands and transmits these signals to the inside of the cell. The Notch pathway consists of Notch and its ligands, as well as intracellular proteins that transmit the Notch signal to the nucleus. Included in the pathway are transcription factors, the proteins that bear the effector function of the pathway.

The effect of Notch signaling is the phenomenon of lateral inhibition, the singling out of one cell from a cell cluster for a given fate (neurogenesis for example), and the inhibition of those cells not elected to differentiate. Lateral inhibition occurs repeatedly in Drosophila development. Central to this process is the interaction of Notch receptor with a group of ligands including Delta, Scabrous and Serrate. Some of these ligands float free around the outside of the cell and others are bound to the surface of cells.

The interaction of Notch with its ligand triggers a chain of intracellular events resulting in lateral inhibition, the prevention of a specified fate committment on the part of most of the cells of a cluster. The first link in the chain of events is a reduction of the affinity of Suppressor of Hairless protein for the cytoplasmic tail of the Notch receptor. Following this, Su(H) is free to enter the nucleus where it assumes its role as a transcription factor. Su(H) mediates transcriptional activation of Enhancer of split complex of genes. This complex will then inhibit the proneural genes achaete, scute, lethal of scute and asense. The transcriptional inhibitory capacity of Enhancer of split complex genes results in lateral inhibition.

The process of lateral inhibition often involves cell migration. In addition to the signaling capacity of Notch receptor, a second important function of Notch and its ligand Delta is that of cell adhesion. Delta is bound to the surface of cells. The Delta-Notch interaction results in an intimate adhesive contact between fated cell, which often migrates, and inhibited cell, bearing Notch, which remains in its germ layer or cluster of origin. Thus the interaction of Notch with its ligand causes the lateral inhibition of the cell bearing Notch, and the migration and triggering of a differentiated state for the cell bearing the Notch ligand. The ligand most often turns out to be Delta.

How can the Notch pathway components be called neurogenic if the function of Notch is to repress the adoption of a differentiated state in the cell that carries Notch protein? There are two answers to this question. The first is that the term neurogenic is a misnomer, arising because the phenotype of Notch pathway mutants manifests the result of an over production of neurons. The pathway is named neurogenic because of this mutant phenotype. The second answer lies in considering the principle ligand of Notch and its function. Delta is not secreted but is cell bound. The Delta-Notch interaction serves a cell adhesive function between ligand and receptor bearing cells. While the receptor bearing cell is inhibited in assuming a differentiated state, the ligand bearing cell is free to do so. In neurogenesis this cell delaminates, and then migrates out of the epithelial cell layer. The ligand bearing cell assumes the differentiated state of neuroblast and physically resides in the developing nervous system. Thus Notch is neurogenic with respect to the cell that bears the Notch ligand.

Developmental biology of the Notch pathway

The Notch ligand receptor system functions in all three germ layers during embryonic development as well as in the germ cell line and in imaginal discs during the larval and pupal stages. The outline below lists developmental processes in which the Notch pathway functions. All are affected by Notch and where tested, all were found to be affected by Delta (Hartenstein, 1992).

The achaete-scute complex

The achaete-scute complex (AS-C) contains four related genes, achaete, scute, lethal of scute and asense. These are spread over 90 kilobases. The proteins coded for by these genes serve as transcription factors. Each has a DNA binding domain, termed a basic helix-loop-helix domain, and three of the four proteins (the exception is Asense) have a C-terminal acidic domain.

achaete and scute genes are more than 25 kb apart. lethal of scute is more than 12 kb from scute. asense is another 45 kb removed from lethal of scute.

These genes are termed proneural, since they promote neuroblast differentiation and are thus essential for the differentiation of the central and peripheral nervous system and the brain. Other functions are also served by these genes: regulation of sex determination (scute); participation in specification of muscle progenitors (lethal of scute), and regulation of sequential fates in Malpighian tubule development (achaete).

How are achaete-scute complex genes activated and inactivated? Genes like hairy, extramachrochaete and pannier help to establish patterns of achaete expression by acting as repressors. The first two genes received their names because of their interaction with achaete: mutation in either produces extra sensory elements such as hairs and machrochaete. As achaete is responsible for activating the sensory cell fate, Hairy and EMC act to restrict achaete transcription to a limited region where sensory elements will form.

NK2/Ventral nervous system defective regulates the expression of achaete and scute to the medial column of the ventral nervous system (Skeath, 1994). Specifically, first NK2 is essential for AS-C gene expression in the medial column of every other neuroblast row through regulatory elements located 3' to achaete, and second, through a 5' regulatory region, NK2 functions to increase or maintain proneural gene expression within the proneural cluster that normally gives rise to the neuroblast (Skeath, 1994).

The above information about the three repressors and NK2 provides only a nearsighted view of activation and repression by the AS-C. There is a more global answer to the question of how AS-C genes are activated. AS-C genes are activated in proneural clusters in the ventral portion of the fly. The anterior-posterior arrangement of neuroepithelial clusters is intimately connected to the process of segmentation. The proneural genes are expressed in each of the 14 segments. Pair rule genes define these segments, and therefore the placement of proneural AS-C clusters. In fact embryos that are mutant in each of the pair rule genes show an alteration of early cluster pattern.

It is not just the presence or absence of a cluster that is controlled by pair rule genes, but the arrangement of neuroblasts within each cluster, and consequently the arrangement of cells expressing AS-C genes. Each cluster is subdivided into subdomains, and each subdomain has its particular arrangement of neuroblasts. Segment polarity genes act here too, especially gooseberry and naked. They help maintain the AP borders of the cells within each cluster (Skeath, 1992).

See Chris Doe's Hyper-Neuroblast map site for information on the origin and lineage of specific neuroblasts in the CNS and for information about the expression of specific genes regulating neuroblast origin.

achaete and scute are controlled by combinations of axis-patterning genes through a common enhancer or locus control region. The pair rule genes act through this region, situated in the intervening chromosomal segment between achaete and scute. Thus this region is under the initial control of products of the pair rule genes and is later maintained by selected segment polarity genes.

Consider the amazing patterning present in insect epidermis. Flies have hairs that appear as reproducable patterns from one individual to another. These patterns are species specific and therefore distinguish one species from another. How are these patterns made?

Further analysis of the 90 kD region between achaete and asense has defined nine site-specific enhancer-like elements that function to regulate achaete and scute expression in proneural clusters of imaginal discs. Ultimately it is the capacity of site specific enhancers to kindle AS-C transcription at very localized regions in the proneural clusters of imaginal discs that is responsible for the patterning of sensory elements seen in adults.

Each of these enhancers, regulated by a specific set of transcription factors defines a prepattern for achaete and scute expression. The enhancers are named after the sensilla in which they direct achaete and scute expression. For example the L3/TSM enhancer, just upstream of scute directs achaete and scute to third vein sensilla campaniforma (L3) and twin sensilla of the anterior wing margin (TSM) (Gomez-Skarmeta, 1995). It is rather remarkable that each sensory organ or group is specificied by a special enhancer selected through evolution for just that purpose. For a historical view of this incredible work see Ghysen, 1988.

Embryonic central nervous system lineages

In Drosophila, central nervous system (CNS) formation starts with the delamination from the neuroectoderm of about 30 neuroblasts (NBs) per hemisegment. These give rise to approximately 350 neurons and 30 glial cells during embryonic development. Understanding the mechanisms leading to cell fate specification and differentiation in the CNS requires the identification of the NB lineages. Each segment is also subdivided along its dorsal-ventral axis. The delamination of the neuroblasts (NBs) from the neuroectoderm occurs between embryonic stages 8 and 11 and is divided into five phases (S1-S5). On each side of the ventral midline there are three longitudinal columns of cells: medial, intermediate and lateral. S1-S3 NBs form these three longitudinal columns. The positions of these three columns is regulated by genes defining the dorsal-ventral axis: twist, snail decapentaplegic and tolloid. These genes likewise act through the enhancer located between achaete and scute (Skeath, 1992). S4 and S5 NBs become interspersed between the existing columns of NBs.

Perpendicular to the columns, NBs are numbered in each segment from anterior to posterior as rows 1-7. Thus NB 1-2 is in the medial column (1) and represents the 2nd neuroblast in each segment. There are a total of thirty identified neuroblasts in each (right or left) half of a segment (hemisegment), and they give rise to a total of 350 progeny cells. For instance, NB 1-1 delaminates preferentially as a S1 NB. Around mid stage 11 the anterior-posterior position of NB1-1 is in line with, or slightly anterior to, the tracheal pits. At this stage it has given rise to a cluster of six to eight daughter cells on its dorsal side. At the beginning of germ band shortening (end stage 11) two of the progeny, the aCC and pCC leave the cluster and move anteriorly. They are the first progeny of NB 1-1. The aCC neuron innervates a dorsal muscle. Each thoracic NB 1-1 gives rise to 8 - 14 cells including ipsilaterally projecting interneurons and 1 or 2 motoneurons. In the abdomen NB 1-1 gives rise to a smaller cluster comprising four to six ipsilaterally projecting interneurons and no motoneurons; in addition the abdominal clone comprises 3 subperineural glial cells (Bossing, 1996). Different segmental fates of each neuroblast is determined by homeotic genes.

Thirteen lineages derived from the dorsal (lateral) part of the neuroectoderm are described here and 12 of them are assigned to identified NBs. Together, the 13 lineages comprise approximately 120 neurons and 22 to 27 glial cells which have been included in a systematic terminology. Therefore, NBs from the dorsal neuroectoderm produce about 90% of the glial cells in the embryonic ventral ganglion. Two of the NBs give rise to glial progeny exclusively (NB 6-4A, GP); five NBs give rise to glia as well as neurons (NBs 1-3, 2-5, 5-6, 6-4T, 7-4). These seven NBs are arranged as a group in the most lateral region of the NB layer. The other lineages (NBs 2-4, 3-3, 3-5, 4-3, 4-4, 5-4, clone y) are composed exclusively of neurons (interneurons, motoneurons, or both). It has been possible to link the lateral cluster of even-skipped expressing cells (EL) to the lineage of NB 3-3. Along with the previously described clones, the vast majority (more than 90%) of cell lineages in the embryonic ventral nerve cord (in the thorax and abdomen) are now known. Previously identified neurons and most glial cells are now linked to certain lineages and, thus, to particular NBs. This complete set of data provides a foundation for the interpretation of mutant phenotypes and for future investigations on cell fate specification and differentiation (Schmidt, 1997).

Aging neural progenitors lose competence to respond to mitogenic Notch signaling

Drosophila neural stem cells (neuroblasts) are a powerful model system for investigating stem cell self-renewal, specification of temporal identity, and progressive restriction in competence. Notch signaling is a conserved cue that is an important determinant of cell fate in many contexts across animal development; for example, mammalian T cell differentiation in the thymus and neuroblast specification in Drosophila are both regulated by Notch signaling. However, Notch also functions as a mitogen, and constitutive Notch signaling potentiates T cell leukemia as well as Drosophila neuroblast tumors. While the role of Notch signaling has been studied in these and other cell types, it remains unclear how stem cells and progenitors change competence to respond to Notch over time. Notch is required in type II neuroblasts for normal development of their transit amplifying progeny, intermediate neural progenitors (INPs). This study finds that aging INPs lose competence to respond to constitutively active Notch signaling. Moreover, reducing the levels of the old INP temporal transcription factor Eyeless/Pax6 allows Notch signaling to promote the de-differentiation of INP progeny into ectopic INPs, thereby creating a proliferative mass of ectopic progenitors in the brain. These findings provide a new system for studying progenitor competence and identify a novel role for the conserved transcription factor Eyeless/Pax6 in blocking Notch signaling during development (Farnsworth, 2015).

BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

Notch signaling plays a crucial role in the control of proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene (see Drosophila Blos2), is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1 related complex. Bloc1s2-/- mice are embryonic lethal and exhibit defects in cortical development and hematopoiesis. Loss of BLOS2 results in elevated Notch signaling, which consequently increases the proliferation of neural progenitor cells and inhibits neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice leads to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacts with Notch1 in endo-lysosomal trafficking of Notch1. These findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking by controlling multiple stem and progenitor cell homeostasis in vertebrates (Zhou, 2016).

Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis

Fine-tuned Notch and Hedgehog signalling pathways via attenuators and dampers have long been recognized as important mechanisms to ensure the proper size and differentiation of many organs and tissues. This notion is further supported by identification of mutations in these pathways in human cancer cells. However, although it is common that the Notch and Hedgehog pathways influence growth and patterning within the same organ through the establishment of organizing regions, the cross-talk between these two pathways and how the distinct organizing activities are integrated during growth is poorly understood. An unbiased genetic screen in the Drosophila melanogaster eye has found that tumour-like growth was provoked by cooperation between the microRNA miR-7 and the Notch pathway. Surprisingly, the molecular basis of this cooperation between miR-7 and Notch converged on the silencing of Hedgehog signalling. In mechanistic terms, miR-7 silenced the interference hedgehog (ihog) Hedgehog receptor, while Notch repressed expression of the brother of ihog (boi) Hedgehog receptor. Tumourigenesis was induced co-operatively following Notch activation and reduced Hedgehog signalling, either via overexpression of the microRNA or through specific down-regulation of ihog, hedgehog, smoothened, or cubitus interruptus or via overexpression of the cubitus interruptus repressor form. Conversely, increasing Hedgehog signalling prevented eye overgrowth induced by the microRNA and Notch pathway. Further, it was shown that blocking Hh signal transduction in clones of cells mutant for smoothened also enhance the organizing activity and growth by Delta-Notch signalling in the wing primordium. Together, these findings uncover a hitherto unsuspected tumour suppressor role for the Hedgehog signalling and reveal an unanticipated cooperative antagonism between two pathways extensively used in growth control and cancer (Da Ros, 2013).

A challenge to understand oncogenesis produced by pleiotropic signalling pathways, such as Notch, Hh, and Wnts, is to unveil the complex cross-talk, cooperation, and antagonism of these signalling pathways in the appropriate contexts. Studies in flies, mice, and in human cell cultures have provided critical insights into the contribution of Notch to tumourigenesis. These studies highlighted that Notch when acting as an oncogene needs additional mutations or genes to initiate tumourigenesis and for tumour progression, identifying several determinants for such co-operation. The identification of these co-operative events has often been knowledge-driven, although unbiased genetic screens also identified known unanticipated tumour-suppressor functions. In this sense, this study describes a conserved microRNA that cooperates with Notch-induced overproliferation and tumour-like overgrowth in the D. melanogaster eye, miR-7. Alterations in microRNAs have been implicated in the initiation or progression of human cancers, although such roles of microRNAs have rarely been demonstrated in vivo. In addition, by identifying and validating functionally relevant targets of miR-7 in tumourigenesis, this study also exposed a hitherto unsuspected tumour suppressor role for the Hh signalling pathway in the context of the oncogenic Notch pathway. Given the conservation of the Notch and Hh pathways, and the recurrent alteration of microRNAs in human cancers, it is speculated that the genetic configuration of miR-7, Notch, and Hh is likely to participate in the development of certain human tumours (Da Ros, 2013).

In human cancer cells, miR-7 has been postulated to have an oncogene or a tumour suppressor functions that may reflect the participation of the microRNA in distinct pathways, due to the regulation of discrete target genes in different cell types, such as Fos, IRS-2, EGFR, Raf-1, CD98, IGFR1, bcl-2, PI3K/AKT, and YY1 in humans (Da Ros, 2013).

In Drosophila, multiple, cell-specific, targets for miR-7 have been previously validated via luciferase or in vivo eGFP-reporter sensors or less extensively via functional studiest. Although microRNAs are thought to regulate multiple target genes, when tested in vivo it is a subset or a given target that predominates in a given cellular context. Indeed, of the 39 predicted miR-7 target genes tested by direct RNAi, only downregulating ihog with several RNAi transgenes (UAS-ihog-IR) fully mimicked the effect of miR-7 overexpression in the transformation of Dl-induced mild overgrowth into severe overgrowth and even tumour-like growth. Moreover, it was confirmed that endogenous ihog is directly silenced by miR-7 and that this silencing involves direct binding of the microRNA to sequences in the 3'UTR of ihog both in vivo and in vitro (Da Ros, 2013).

Nevertheless, other miR-7 target genes may contribute to the cooperation with Dl-Notch pathway along with ihog, such as hairy and Tom. While miR-7 can directly silence hairy in the wing, this effect has been shown to be very modest, and thus, it is considered that while hairy may contribute to such effects, it is unlikely to be instrumental in this tumour model. Indeed, the loss of hairy is inconsequential in eye development, although retinal differentiation is accelerated by genetic mosaicism of loss of hairy and extramacrochaetae that negatively sets the pace of MF progression. It is unclear how Hairy might contribute to Dl-induced tumourigenesis (Da Ros, 2013).

The RNAi against Tom produced overgrowth with the gain of Dl albeit inconsistently and with weak penetrance, where one RNAi line did not modify the Dl-induced overgrowth and the other RNAi line caused tumours in less than 40% of the progeny. Tom is required to counteract the activity of the ubiquitin ligase Neuralized in regulating the Notch extracellular domain, and Dl in the signal emitting cells. These interactions are normally required to activate Notch signalling in the receiving cells through lateral inhibition and cell fate allocation. However, although it remains to be shown whether similar interactions are active during cell proliferation and growth, the moderate enhancement of Dl that is induced when Tom is downregulated by RNAi suggests that miR-7-mediated repression of Tom may contribute to the oncogenic effects of miR-7 in the context of Dl gain of function, along with other targets such as ihog (Da Ros, 2013).

Conversely, while the target genes of the Notch pathway, E(spl)m3 and E(spl)m4 as well as E(spl)mγ, Bob, E(spl)m5, and E(spl)mδ, have been identified as direct targets of miR-7 in the normal wing disc via analysis of 3'UTR sensors, there was no evidence that HLHm3, HLHm4, HLHm5, Bob, and HLHmγ are biological relevant targets of miR-7 in the Dl overexpression context. HLHmδ RNAi produced inconsistent phenotypes in the two RNAi transgenic lines available, causing tumour-like growth at very low frequency in only one of the lines. No evidence was obtained that miR-7 provoked overgrowth by targeting the ETS transcription factor in the EGFR pathway AOP/Yan, a functionally validated target of the microRNA miR-7 during retinal differentiation. Neither was any evidence obtained that RNAi of atonal provoked eye tumours with Dl overexpression, although a strong inhibition via expression of a fusion protein Atonal::EN that converts Atonal into a transcriptional repressor has been shown to be sufficient to trigger tumorigenesis together with Dl. Thus, it was reasoned that given that microRNA influenced target genes only subtly (even when using ectopic expression), it is possible that downregulation of atonal contributes to the phenotype along with the other targets (Da Ros, 2013).

In conclusion, this study has identified cooperation between the microRNA miR-7 and Notch in the D. melanogaster eye and identified and validated ihog as a direct target of the miR-7 in this context and have identified boi as a target of Notch-mediated activity at the DV eye organizer, although it remains whether this regulation is direct or indirect. A hitherto unanticipated tumour suppressor activity was uncovered of the endogenous Hh signalling pathway in the context of gain of Dl-Notch signalling that is also apparent during wing development (Da Ros, 2013).

Hh tumour suppressor role is revealed when components of the Hh pathway were lost in conjunction with a gain of Dl expression in both the eye and wing discs. Hh and Notch establish signalling centres along the AP and DV axes, respectively, of the disc to organize global growth and patterning. Where the organizer domains meet, the Hh and Notch conjoined activities specify the position of the MF in the eye disc and the proximodistal patterning in the wing disc. This study also unvailed that in addition antagonistic interaction between the Hh and Notch signalling might help to ensure correct disc growth. Thus, it was shown that Hh signalling limits the organizing activity of Dl-Notch signalling. Although it is often confounded whether Dl-Notch signalling instructs overgrowth by autonomous or nonautonomous (i.e., DV organizers) mechanisms, these findings uncover that loss of Hh signalling enhances a non-cell autonomous oncogenic role of Dl-Notch pathway (Da Ros, 2013).

To date, Hh has not yet to be perceived as a tumour suppressor, although it is noteworthy that human homologs of ihog, CDO, and BOC were initially identified as tumour suppressors. Importantly, both CDO and BOC are downregulated by RAS oncogenes in transformed cells and their overexpression can inhibit tumour cell growth in vitro. Since human RAS regulates tumourigenesis in the lung by overexpressing miR-7 in an ERK-dependent manner, it is possible that RAS represses CDO and BOC via this microRNA. Indeed, the 3'UTR of both CDO and BOC like Drosophila ihog contains predicted binding sites for miR-7. There is additional clinical and experimental evidence connecting elements of the Hedgehog pathway with tumour-suppression. The function of Growth arrest specific gene 1 (GAS1), a Hh ligand-binding factor, overlaps that of CDO and BOC, while its overexpression inhibits tumour growth . More speculative is the association of some cancer cells with the absence of cilium, a structure absolutely required for Hh signal transduction in vertebrate cells (Da Ros, 2013).

Given the pleiotropic nature of Notch, Wnts, BMP/TGFβ, Ras, and Hh signalling pathways in normal development in vivo, it is speculated that competitive interplay as that described in this study between Notch and Hh may not be uncommon among core growth control and cancer pathways that act within the same cells at the same or different time to exert multiple outputs (such as growth and cell differentiation). Moreover, context-dependent tumour suppressor roles could explain the recurrent, unexplained, identification of somatic mutations in Hh pathway in human cancer samples. Indeed, the current findings stimulate a re-evaluation of the signalling pathways previously considered to be exclusively oncogenic, such as the Hh pathway (Da Ros, 2013).

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. This study shows that Notch regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, this study shows that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. These results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology (Wu, 2017).

By revealing the involvement of the miRNA pathway, this study highlights the complexity of the N signaling network in normal NSCs and tumor-forming cancer stem cell (CSC)-like NSCs. Previous studies implicated critical roles for both canonical and non-canonical N signaling pathways in NSCs and CSC-like NSCs, and revealed particular dependence of CSC-like NB growth on non-canonical N signaling, which involves PINK1, mTORC2, and mitochondrial quality control. The current study reveals a particular requirement for ban in CSC-like NBs induced by N hyperactivation. The CSC-like NB overproliferation induced by hyperactivation of N or N pathway component Dpn can all be assumed to be of type II NB origin, since previous studies have clearly established that Notch signaling is essential for the development and/or maintenance of type II NBs, but dispensable for type I NBs, and that hyperactivation of Notch or its downstream effector Dpn induced ectopic CSC-like NB growth by altering the lineage homeostasis of the type II but not type I NBs. It would be interesting to test whether, in addition to ban's role in canonical N signaling, there exists a link between ban and non-canonical N signaling. The data indicate that the ban-Numb signaling motif regulates NSC/CSC behavior through at least two mechanisms. On one hand, it regulates cell growth and particularly nucleolar growth, through Myc, a known regulator of cellular and nucleolar growth. Consistently, negative regulation of Myc protein level by Numb was observed through E3 ubiquitin-protein ligase, Huwe1, and the UPS. c-Myc is an essential regulator of embryonic stem cell (ESC) self-renewal and cellular reprogramming, and Myc level and stability can be controlled in stem cells through targeted degradation by the UPS, suggesting conserved mechanisms. A key function of the nucleolus is the biogenesis of ribosomes, the cellular machinery for mRNA translation, and previous studies in Drosophila have supported the critical role of nucleolar growth in NSC self-renewal and maintenance. On the other hand, the ban-Numb axis feedback regulates the activity of N by a double negative regulation, with the end result being positive feedback regulation. This feedback mechanism may help transform initial not so dramatic differences in N activity between NB and its daughter cell generated by the asymmetric segregation of Numb during NB division [33] into 'all-or-none' decision of cell fates. Feed-forward regulatory loops, both coherent and incoherent, are frequently found in gene regulatory networks, and although ban miRNA is not conserved in mammals, miRNAs have been implicated in an incoherent feed-forward loop in the Numb/Notch signaling network in colon CSCs in mammals (Wu, 2017).

Given the role of ban in a positive feedback regulation of N and the potency of N hyperactivity in inducing tumorigenesis, one may wonder why ban overexpression is not sufficient to cause tumorigenesis. As in any biological systems, feedback regulation is meant to increase the robustness and maintain homeostasis of a pathway. Feedback alone, either negative or positive, should not override the main effect of the signaling pathway. Thus, in the NB system feedback regulation by ban is built on top of the available N signaling activity in a given cell and serving to maintain N activity. Because of ban's 'fine-tuning' rather than 'on/off switching' of Numb expression, its effect on N activity during feedback regulation will also be 'fine-tuning', serving to maintain N activity in NB within a certain range. Overexpression of ban in a wild type background may not be sufficient to cause tumorigenesis because N activity is not be elevated to the level sufficient to induce brain tumor as in N-v5 overexpression condition. Consistent with this, the extent of Numb inhibition by ban is also modest, not reaching the threshold level of Numb inhibition needed to cause tumorigenesis. Consistent with the notion that feedback regulation by ban is built on top of the available N signaling activity in a given cell, and that there is dosage effect of N activity in tumorigenesis, overexpression of ban in N-v5 overexpression background further enhanced N-v5 induced tumorigenesis. It is likely that ban or other miRNAs may participate in additional regulatory mechanisms in the N signaling network in Drosophila. Of particular interest, it would be interesting to test whether miRNAs may impinge on the asymmetric cell division machinery to influence the symmetric vs. asymmetric division pattern, a key mechanism employed by NSCs and transit-amplifying IPs to balance self-renewal with differentiation (Wu, 2017).

The results emphasize the critical role of translational control mechanisms in NSCs and CSC-like NSCs. Compared to the heavily studied transcriptional control, knowledge of the translational control of NSCs and CSCs is rather limited. As fundamental regulators of mRNA translation, miRNAs can interact with both positive and negative regulators of translation to influence gene expression. Thus, miRNA activity can be regulated context-dependently at both the transcriptional and translational levels, which may account for the opposite effect of N on ban activity in the fly brain and wing disc, although the ban genomic locus is bound by Su(H) in both tissues. Whether N regulates the transcription of ban or its activity as a translational repressor in the wing disc remains to be tested. With regard to the translation of numb mRNA, the conserved RNA-binding protein (RNA-BP) Musashi has been shown to critically regulate the level of Numb protein in mammalian hematopoietic SCs and leukemia SCs. Further investigation into the potential interplay between miRNAs and RNA-BPs in the translational control of Numb in NBs and CSC-like NBs promises to reveal new mechanisms and logic in stem cell homeostasis regulation, with important implications for stem cell biology and cancer biology (Wu, 2017).

Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam

Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. The large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, this study focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. It is concluded that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, it is proposed that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. The data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. It is proposed that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors (Panchal, 2017).

Stem cells retain the capacity for development in differentiated organisms, which is important for tissue growth, homeostasis and regeneration, and for long-term reproductive capability. Stem cells are often associated with a specialized microenvironment, a niche that is essential for the formation, maintenance, and self-renewal of stem cells by preventing cell differentiation and controlling rate and mode of cell division. The niche for the germline stem cells (GSCs) in Drosophila serves as an important model for the analysis of interactions between niche and stem cells. The astounding fecundity of Drosophila females that can lay dozens of eggs per day over several weeks depends on approximately 100 GSCs that are sustained by 40 stem cell niches. To understand the formation and maintenance of these GSCs, it is important to understand how stem cell niches form and how they function (Panchal, 2017).

The GSC niche of the Drosophila ovary consists of three somatic cell types: cap cells, escort cells, and terminal filament (TF) cells. GSCs are anchored to cap cells by DE-cadherin-mediated adhesion and require close proximity to cap cells to retain stem cell character. Cap cells secrete the BMP homolog Decapentaplegic (Dpp), activating the TGFβ signaling pathway in adjacent GSCs, which leads to the repression of the germline differentiation factor Bag-of-Marbles (Bam). Through Hedgehog (Hh) signaling, cap cells also appear to stimulate escort cells to secrete Dpp. The combined pool of Dpp from cap and escort cells, together with mechanisms that concentrate Dpp in the extracellular space around GSCs, promotes the maintenance of 2-3 GSCs, whereas the adjacent GSC daughter cells that have lost the contact to cap cells will enter differentiation as cystoblasts. In contrast, TFs are not in direct contact with GSCs but serve important functions in the development and probably also in the maintenance and function of GSC niches (Panchal, 2017).

Formation of GSC niches begins with the progressive assembly of TFs by cell intercalation during the 3rd larval instar. The process of TF cell specification is not understood but might start in 2nd instar when the first TF precursor cells appear to leave the cell cycle. TF morphogenesis depends on the Bric à brac transcriptional regulators that control the differentiation of TF cells and their ability to form cell stacks, and involves the Ecdysone Receptor (EcR), Engrailed, Cofilin, and Ran-binding protein M (RanBPM). The number of TFs that form at the larval stage determine the number of GSC niches at the adult stage, and are regulated by several signaling pathways that control cell division and timing of cell differentiation in the larval ovary, including the EcR, Hippo and Jak/Stat, Insulin and Activin pathways. Despite the recent advance in elucidating mechanisms that control the number of GSC niches and the temporal window in which they form, relatively little is known about the origin and specification of the somatic cell types of the GSC niche (Panchal, 2017).

Notably, the origin and specification of cap cells, the main component of an active GSC niche is little understood. Cap cells (also called germarial tip cells) are first seen at the base of completed TFs at the transition from the 3rd larval instar to prepupal stage. They appear to derive from the interstitial cells (also called intermingled cells) of the larval ovary that are maintained by Hh signaling from TFs. The formation of cap cells is accompanied by the establishment of GSCs. The N pathway contributes to the development of cap cells. A strongly increased number of functionally active cap cells per niche form in response to overexpression of the N ligand Delta (Dl) in germline or somatic cells, or the constitutive activation of N in somatic gonadal cells. The ability of N to induce additional cap cells seems to depend on EcR signaling. Loss of Dl or N in the germline had no effect on cap cells. However, loss of N in cap cell progenitors or Dl in TF cells caused a decrease in the number of cap cells. A current model suggests that Dl signaling from basal-most TF cells to adjacent somatic cells together with Dl signaling between cap cells allows for a full complement of cap cells to form. Furthermore, N protects cap cells from age-dependent loss as long as its activity is maintained by the Insulin receptor. The Jak/Stat pathway, which operates downstream or in parallel to the N pathway in the niche, is not required for cap cell formation. As cap cells were reduced in number but never completely missing when the N pathway components were compromised, the question remains whether N signaling is the only factor that is important for cap cell formation. Furthermore, no factor that operates downstream of N has been identified that is crucial for cap cell formation (Panchal, 2017).

This study finds that Traffic jam (Tj) is both required for cap cell specification and for the morphogenetic behavior of cap cells, enabling them to form a properly organized niche that can accommodate 2-3 GSCs. Tj is a large Maf transcription factor that belongs to the bZip protein family. Its four mammalian homologs control differentiation of several cell types and are associated with various forms of cancer. Tj is essential for normal ovary and testis development, and is only expressed in somatic cells of the gonad. Interestingly, Tj is present in cap cells and escort cells but not in TFs. This study shows that Tj is essential for the formation of the GSC niche. First, Tj regulates the behavior of cap cells, enabling them to form a cell cluster instead of a cell stack, which appears to be important for the formation of a normal-sized GSC niche with the capacity to support more than one GSC. Second, cap cells adopt the fate of TF cells in the absence of Tj function, and TF cells develop cap cell-like features when forced to express Tj, indicating that Tj specifies the cap cell fate. Genetic interactions suggest that Tj and N are required together for cap cell formation, but have different functions in this process. For somatic gonadal cells to adopt the cap cell fate, it is proposed that Tj has to be present to inhibit the TF cell fate and N has to be present to prevent the escort cell fate and/or produce the correct number of cap cell precursors (Panchal, 2017).

Loss of Tj has a profound negative effect on the establishment, number, and maintenance of GSCs. Effects of Tj on the germline were previously shown to be indirect as Tj is neither expressed nor cell-autonomously required in the germline. Therefore, it is proposed that the dramatic change in the structure of the somatic niche affects GSCs when Tj function is compromised. An inverse causal relationship, where a reduced number of GSCs would trigger the somatic niche defects was ruled out by showing that cap cells can still look and behave normally in the absence of any germ cells. It is concluded that Tj controls GSCs indirectly by controlling somatic cell fate and cell arrangement in the stem cell niche (Panchal, 2017).

By controlling the morphology and behavior of the cap cells, Tj regulates the GSC-carrying capacity of the niche. When Tj expression was moderately reduced, the number of GSCs per niche was reduced, with the remaining GSC properly maintained over several weeks. The decrease of GSCs per niche correlated with a decrease of cap cells in the germarium. Two cap cells were on average required to sustain one GSC, similar to what has been proposed for a wild-type ovary. The data indicate that the reduced niche capacity is due to a reduction in the available contact surface between cap cells and GSCs. Tj-depleted cap cells that convert from forming a cluster inside the germarium to forming a stalk outside the germarium minimize their availability for GSC attachment. A connection between the GSC-cap cell contact area and niche capacity is similarly reflected in the increased number of GSCs that accompanies an increase in cap cell size due to loss of RanBPM. This study shows that the spatial arrangement of the cap cells has a crucial impact on the number of stem cells per niche (Panchal, 2017).

When Tj function was completely abolished, the number of GSCs was drastically reduced, as expected in the absence of cap cells. The very few pMad-positive GSC-like cells in tj mutant prepupal ovaries were always associated with a TF, suggesting that TFs might temporarily provide enough Dpp to activate Mad in a few germline cells, consistent with the finding that Dpp is expressed in TFs at the late larval stage\. This is not sufficient, however, to maintain GSCs and adult ovaries rarely contain pMad-positive germline cells. This is in agreement with the finding that Dpp is not detected in adult TFs, and corroborates that cap cells are required for GSC maintenance. In addition, the rapid loss of the entire germ cell pool in Tj-depleted ovaries during the pupal stage might be precipitated by loss or defects in escort cells. Escort cell precursors are not properly intermingled with germ cells at the larval stage and differentiated escort cells appear to be missing in adult ovaries that lack Tj. As escort cells are crucial for germ cell differentiation, the defect in escort cell differentiation could be responsible for the demise of the germline in tj mutants (Panchal, 2017).

GSCs have broad cellular protrusions, which they use to reach and tightly ensheath the accessible surface of cap cells. In wild type, relatively short protrusions are sufficient to make extensive contact with more than one cap cell. However, when cap cells formed a stalk, GSCs were often observed to produce unusually long extensions that allowed them not only to contact the immediate cap cell neighbor but also a more distantly located cap cell. This suggests that GSCs respond to a chemotactic signal from cap cells and send protrusions toward this signal. It remains to be investigated whether this is a response to Dpp signaling or signaling through another pathway. The importance of cellular protrusions in signaling events in the stem cell niche has recently come to light with the discovery of nanotubes that mediate Dpp signaling between GSCs and hub cells in the Drosophila testes, and cytonemes that contribute to Hh signaling from cap to escort cells in the ovary (Panchal, 2017).

This analysis shows that Tj is required for the specification of cap cells. In the absence of Tj function, additional TF cells form at the expense of cap cells, resulting in unusually long TFs while the cap cell fate is not established. Whereas the formation of cap cell precursors appears not to require Tj, this transcription factor is essential for the ability of these precursors to take on the cap cell fate and to prevent the TF cell fate that is otherwise adopted as a default state. The following findings support this conclusion: (1) In the absence of Tj function, cap cells were missing while additional cells that displayed TF cell-characteristic morphology, behavior and marker expression were integrated into the TF. The number of additional TF cells was comparable to the normal number of cap cells. (2) Prospective cap cells cell-autonomously adopted a TF-specific morphology and behavior in the absence of functional Tj. (3) A hypomorphic tj mutant provided direct evidence for the incorporation of cap cells into TFs, forming the basal portion of these stalks. (4) Ectopic expression of Tj in TF cells caused a change toward cap cell-typical marker expression and morphology. Together, these data demonstrate that Tj promotes cap cell specification (Panchal, 2017).

The expression pattern of Tj supports the notion that Tj has a function in cap cells but not in TF cells. Tj is continuously expressed in cap cells. Tj is also present in the anterior interstitial cells of the larval ovary, which are thought to develop into cap cells. In contrast, Tj is neither detected in the cell population that gives rise to TFs during 3rd larval instar, nor in differentiated TFs. Interestingly, even in the absence of Tj function, the tj gene remains differentially expressed in the anterior niche, being inactive in regular TF cells but active in the additional TF cells, which form the apical and basal portion of a TF, respectively. This differential expression of Tj indicates that a regionally or temporally regulated mechanism operates upstream of Tj that initiates differences in anterior niche cells. Although it is conspicuous that Tj expression from 3rd instar onwards is restricted to cells that are in direct contact with germline cells, which includes cap cells but excludes TF cells, it has previously been shown that Tj expression is not dependent on the germline. This suggests that a soma-specific mechanism is responsible for the differential expression of Tj in anterior niche cells. Interestingly, a recent study uncovered the importance of Hh signaling from TFs to neighboring interstitial cells in the larval ovary and proposes that tj is a direct target of the Hh signaling pathway (Panchal, 2017).

The current findings suggest the presence of a new cell type in the GSC niche that has been named 'transition cell' as it is located between the cap cell cluster and the TF, connecting these two structures of the niche. Notably, the one or occasionally two transition cells have the morphology of TF cells and align with neighboring TF cells despite displaying a cap cell-like marker profile that includes the expression of Tj—although Tj expression is substantially lower than in cap cells. Interestingly, cap cells from ovaries with reduced Tj expression (tjhypo) similarly displayed a TF cell-like morphology and behavior while their expression profile remained cap cell-like. A similar, although weaker effect was noted in a tj hemizygous condition, suggesting that Tj function is haplo-insufficient in cap cells. Thus, when Tj levels are reduced, cap cells adopt very similar molecular and morphogenetic properties as the transition cell in a wild-type niche, and might have adopted this cell fate (Panchal, 2017).

Together, the current findings indicate that Tj has an important role in the establishment of three cell types in the GSC niche: TF cells, transition cells, and cap cells. As lack of Tj function seems to cause a transformation of cap and transition cells into TF cells, and a mild reduction of Tj a cap to transition cell transformation, it is proposed that different Tj expression levels establish different cell fates and morphogenetic traits. It is proposed that a high concentration of Tj leads to the formation of cap cells and a lower concentration to the formation of the transition cell, whereas absence of Tj is required for the formation of TF cells. This model implies that different levels of Tj have different effects on target genes. It is predicted that Tj has at least one target gene that only responds to high levels of Tj and that specifically controls the morphogenetic behavior of cap cells, allowing them to adopt a round morphology and organize into a cell cluster. Whether this relates to an effect of Tj on the expression of adhesion molecules as observed in other gonadal tissues awaits further analysis (Panchal, 2017).

This study identifies Tj as essential for cap cell formation. In addition, this process depends on the N pathway. Therefore, it was asked how the functions of Tj and N in cap cell formation relate to each other. A comparison between the loss and gain-of-function phenotypes suggests that Tj and N have different functions in the establishment of cap cells. In the absence of Tj function, cap cell precursor cells are present but take on the fate of TF cells, whereas depletion of N leads to a loss of cap cells but does not cause the formation of additional TF cells. Ectopic activation of N can induce a strong increase in the number of cap cells, whereas overexpression of Tj did not appear to affect the number of cap cells. Therefore, both factors are important for cap cell formation but contribute differently to this process. The questions then are: What is the respective contribution of Tj and N to cap cell formation, and how are their functions related (Panchal, 2017)?

The function of N in cap cell formation is still not fully understood. The observation that depletion of N reduces the number of cap cells confirms previous findings. However, neither in this nor any previously published experiments were cap cells lost completely when the N pathway was compromised, and it remains therefore unclear whether N is de facto essential for cap cell formation or primarily functions in regulating the size of the cap cell pool. Interestingly, evidence amounts to a function of the N pathway in a decision between the cap cell and escort cell fate: First, Dl signal from TF cells activates the N pathway in adjacent interstitial cells, inducing them as cap cells, whereas the remaining interstitial cells are thought to develop into escort cells. Second, escort cells expressing activated N can develop into cap cells. Third, when tj-Gal4 was used to express active N in interstitial cells, the number of cap cells dramatically increased while the escort cell region became smaller, and some germaria seemed to lack escort cells all together. These germaria also lacked germline cells, although a larger pool of cap cells was expected to increase the number of GSCs. However, the absence of germline cells is consistent with an absence of escort cells, as escort cells have been shown to be important for maintaining the germline. Together, these observations support the hypothesis that N is involved in a cap cell versus escort cell fate decision, and suggest that the N pathway might promote the formation of cap cells by inhibiting the escort cell fate (Panchal, 2017).

To determine how the functions of Tj and N depend on each other, genetic interactions were examined. The N pathway seems to be still functional in tj mutants. First, the expression of N and Dl appeared unaffected and E(spl) was activated in the additional TF cells (= transformed cap cells) similarly to normal cap cells. Second, the formation of additional TF cells in the absence of Tj depended on the presence of N, as only very few additional TF cells formed in a N compromised background. These findings indicate that the N pathway is still active in cap cell precursors when Tj function is abolished. This together with the observation that constitutively active N cannot suppress the tj mutant phenotype suggests that Tj does not act upstream of N in regulating cap cell fate (Panchal, 2017).

Therefore, it was asked whether Tj might operate downstream of N. Loss of Tj was not detected upon N depletion, and this together with the finding that Tj is expressed in all interstitial cells, and not only in those that receive Dl signaling argues against a requirement of N signaling for tj expression. If at all, one would expect tj to be negatively regulated by N as cap cells express a lower level of Tj than escort cells. The maintenance of somatic cell types in N mutant ovaries that are lost in tj mutant ovaries, including the escort cells is also not consistent with a linear relationship. Nevertheless, the ability of Tj to promote the formation of cap cells appears to depend on the activity of the N pathway in cap cell precursors. Again, this is suggested by the finding that when N and Tj were both compromised, the number of additional TF cells were much smaller than when N was fully active. Therefore, it is proposed that N activity sets aside a pool of percursor cells that in the presence of Tj take on the cap cell fate, and in its absence the TF fate (Panchal, 2017).

Similar to the ovary, N is important for the formation of the GSC niche (= hub) in the Drosophila testis. Interestingly, N contributes to hub cell specification by downregulating the expression level of Tj. Not only is the hub still present in tj mutant testes but additionally, ectopic hub cells form in the absence of Tj. Thus, Tj seems to have opposing functions in testes and ovaries, suppressing the niche cell fate in the testis, while promoting it in the ovary (Panchal, 2017).

The interplay between Tj and N seems not restricted to the cap cell fate in the ovary. Whereas neither factor alone is required for TF cell formation, as TF cells formed normally in the absence of either Tj or N, the combined loss of Tj and N led to a strong reduction in the number of TFs and number of TF cells within stalks. This suggests that their combined action is already required at an earlier stage of ovary development, when Tj is still expressed in all somatic cells of the ovary. Moreover, Tj knockdown combined with expression of activated N caused TF cells to be the only cell type remaining of the ovary, indicating that several cell types in the ovary require proper input from both factors. Taken together, the findings support a model, in which both Tj and N operate together to promote the cap cell fate but have separate functions. It is proposed that Tj and N promote the cap cell fate by blocking the TF cell fate and escort cell fate, respectively, and that the combined actions of Tj and the N pathway are required to establish the cap cell fate (Panchal, 2017).

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. This report describes the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. This study shows that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, this study demonstrates that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures (Zhu, 2017).

In contrast to vertebrates that have a single Pax6 gene, the Drosophila genome contains two Pax6 homologs, ey and toy. Both genes are expressed broadly throughout the entire eye-antennal disc but are later limited to a far more restricted domain within the undifferentiated cells of the eye field. Whereas most studies on Pax6 in the eye-antennal disc have focused on the developing compound eye, several reports have hinted at a role for both genes outside of the eye. However, the underlying mechanism of how Ey/Toy promote eye-antennal disc development has been elusive. This is, in part, because of the use of single Pax6 mutants to study development. The phenotypes associated with individual mutants are variable and often restricted to the eye. Several studies have suggested that Ey and Toy function redundantly to each other. This finding most likely explains the variability of phenotype severity and penetrance. Thus, the combined loss of both Ey/Toy may be a more accurate reflection of the effect that Pax6 loss has on Drosophila development. Indeed, this appears to be the case as it is reported that the combined loss of both ey and toy leads to the complete loss of all head structures that are derived from the eye antennal disc. This study attempted to determine the mechanism by which Ey/Toy support eye-antennal disc development (Zhu, 2017).

Previous studies in the fly eye proposed that Pax6 is concerned solely with eye specification, whereas Notch signaling and other retinal determination proteins, such as Eyg, Tsh, and Hth, control cell proliferation and tissue growth. This study proposes an alternate model in which Ey/Toy are in fact required for cell survival and proliferation in addition to eye specification. The data indicate that Ey/Toy regulate growth of the eye-antennal disc through Tsh, N/Eyg, and additional N-dependent proliferation promoting genes. It is proposed that on simultaneous removal of Ey and Toy the eye-antennal disc fails to develop, in part, because the expression of eyg and tsh is lost in complete absence of Pax6. Expression of tsh and activation of the N pathway are sufficient to restore tissue growth to the eye-antennal disc. Support for this model linking Ey/Toy to cell proliferation via Eyg and Tsh comes from studies showing that eyg loss-of-function mutants display a headless phenotype identical to that seen in the ey/toy double knockdowns, that cells lacking eyg do not survive in the eye disc, and overexpression of Tsh causes overproliferation (Zhu, 2017).

The results also show that the combined loss of Ey and Toy affects the number of cells that are in S and M phases of the cell cycle. This observation directly supports the model that Ey/Toy control growth of the eye-antennal disc and is consistent with studies in vertebrates that demonstrate roles for Pax6 in the proliferation of neural progenitors within the brain. Earlier studies observed cells undergoing apoptosis in Pax6 single-mutant eye-antennal discs and showed that blocking cell death alone can partially rescue the head defects of the eyD and toyhdl mutants. Although this study shows that retinal progenitor cells lacking both Pax6 proteins undergo even greater levels of apoptosis, blocking cell death does not restore the eye-antennal disc. What accounts for the differences in the two experiments? In the eyD and toyhdl rescue experiments, each genotype contained wild-type copies of the other Pax6 paralog, but this study has knocked down both Pax6 genes simultaneously. Another possible difference is that Pax6 levels are being reduced while the eyD and toyhdl mutants are likely functioning as dominant negatives. It is concluded from these results that a reduction in cell proliferation but not elevated apoptosis levels is the proximate cause for the complete loss of the eye-antennal disc (Zhu, 2017).

Although the activation of Tsh and the Notch pathway can restore antennal and head epidermal development, neither factor is capable of restoring eye development to the ey/toy double-knockdown discs. This is most likely because both Pax6 genes are also required for the specification of the eye. In particular, Ey/Toy are required for the activation of several other retinal determination genes, including so, eya, and dac. Thus, the results suggest that Notch signaling, Eyg, and Tsh can restore nonocular tissue growth to the eye field but cannot compensate for the Pax6 requirement in eye specification (Zhu, 2017).

Finally, the results using the double knockdown of ey/toy are consistent with the dosage effects that are seen in mammalian Pax6 mutants. Although mutations in ey have just eye defects, the combined loss of ey/toy lacks all head structures. Mice that are heterozygous for Pax6 mutations have small eyes, whereas those that are homozygous completely lack eyes, have severe CNS defects, and die prematurely. Similarly, human patients carrying a single mutant copy of Pax6 suffer from aniridia, whereas newborns that are homozygous for the mutant Pax6 allele have anophthalmia, microcephaly, and die very early as well. As a master control gene of eye development, Pax6 appears to initiate both retinal specification and proliferation. These data demonstrate that the functions of Ey and Toy in the eye-antennal disc are redundant and dependent upon gene dosage, thereby making the roles of Pax6 in the Drosophila similar to what is observed in vertebrates where Pax6 controls both specification and proliferation of the brain and retina in a dosage-sensitive manner (Zhu, 2017).

Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling

Stem cell niches act as signaling platforms that regulate stem cell self-renewal and sustain stem cells throughout life; however, the specific developmental events controlling their assembly are not well understood. This study shows that during Drosophila ovarian germline stem cell niche formation, the status of Notch signaling in the cell can be reprogrammed. This is controlled via steroid-induced miR-125, which targets a negative regulator of Notch signaling, Tom. Thus, miR-125 acts as a spatiotemporal coordinator between paracrine Notch and endocrine steroid signaling. Moreover, a dual security mechanism for Notch signaling activation exists to ensure the robustness of niche assembly. Particularly, stem cell niche cells can be specified either via lateral inhibition, in which a niche cell precursor acquires Notch signal-sending status randomly, or via peripheral induction, whereby Delta is produced by a specific cell. When one mechanism is perturbed due to mutations, developmental defects, or environmental stress, the remaining mechanism ensures that the niche is formed, perhaps abnormal but still functional. This guarantees that the germline stem cells will have their residence, thereby securing progressive oogenesis, thus, organism reproduction (Yatsenko, 2018).

The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking

The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. This study shows that the retromer complex directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7(+) enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. These results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation (Li, 2018).

Unidirectional Notch signaling is a widely used strategy for initiating and maintaining binary cell fates. However, the molecular mechanisms establishing the unidirectionality of Notch signaling in stem cell lineages remain unclear. This study shows that, while asymmetric partition of Numb leads to a biased internalization of the Notch receptor and hence asymmetric dampening of Notch signaling in neural progenitors, it meanwhile poses a high risk of non-canonical endosomal activation of Notch. The retromer complex was found to be the key protein trafficking machinery that resolves this crisis through a timely retrieval of the Notch receptor from its endosomal activation compartments. Upon retromer dysfunction, neural progenitors dedifferentiate into neural stem cell-like status and result in the formation of transplantable tumors. Therefore, retromer acts as a tumor suppressor in Drosophila larval brains. Importantly, mammalian Vps35 physically interacts with Notch, colocalizes with Notch in neural progenitors, and its neuroblast-lineage-specific expression fully rescues neural progenitor-derived brain tumor phenotype in vps35 mutants. Thus, the brain tumor suppressor function of retromer is likely to be conserved in mammals. Intriguingly, downregulation of the retromer complex components has been reported in various human cancers, including glioblastoma. These studies thus provide a new mechanistic link between the retromer complex and carcinogenesis (Li, 2018).

Why the E3 ubiquitin ligase system promoting Notch receptor polyubiquitination and degradation is inherently inefficient in neuroblast lineages? It is speculated that Notch is probably not the only substrate of Su(dx) and Ndfip in neuroblasts or neural progenitors. Therefore, high levels and/or activity of this E3 ubiquitin ligase system above certain threshold may potentially cause imbalanced homeostasis of its critical substrates and hence perturbed neuroblast lineages. Indeed, co-overexpression of Su(dx) and Ndfip led to drastically reduced number of neuroblast lineages and severe tissue atrophy. In this case, a relatively general yet inefficient ubiquitination-degradation system coupled with a highly efficient and selective cargo retrieving system provides a customized regulation of the Notch receptor, ensuring sufficient dampening of Notch signaling in neural progenitors without devastating side effects (Li, 2018).

Intriguingly, previous studies posited that retromer dysfunction causes increased levels of APP (β-amyloid precursor protein) to reside in the endosomes for longer duration than normal, resulting in accelerated processing of APP into amyloid-β, a neurotoxic fragment implicated AD pathogenesis. Furthermore, retromer maintains the integrity of photoreceptors by avoiding persistent accumulation of rhodopsin in endolysosomal compartments that stresses photoreceptors and causes their degeneration. Taken together with this study, these findings indicate that retromer serves as bomb squad to retrieve and disarm harmful or toxic protein fragments from endosomes in a timely manner and thereby safeguard the integrity and fitness of the neuronal lineages (Li, 2018).

How is the Notch receptor ectopically activated in retromer mutants? The idea is favored that Notch is activated in MVBs in a ligand-dependent, cell-autonomous manner, distinct from the majority of non-canonical Notch activation mechanisms. Most of the endosomal Notch activation events identified before, including ectopic Notch signaling activation in ESCRT mutants, BLOS2 mutants, or Rme8 and Vps26 double knockdown background, as well as Hif-alpha-dependent activation of Notch signaling implicated in crystal cell maintenance and survival, are all ligand-independent. It has been proposed that the proteases within the acidifying environment of MVB lumen are sufficient to remove the extracellular domain of Notch, leading to the S3 cleavage of Notch at the limiting membrane. Strongly supporting this notion, blocking the entry of Notch into the ESCRT pathway but not ligand inactivation potently inhibited ectopic Notch activation induced by ESCRT mutations. In sharp contrast to these previously-revealed mechanisms, attenuating ligand activity but not preventing Notch from entering the ESCRT pathway effectively rescues Notch overactivation phenotype caused by retromer dysfunction. Then how Notch signaling is ectopically activated in a ligand-dependent manner in retromer mutants? It is speculated that, upon retromer dysfunction, both Notch and Delta are entrapped in MVBs, where Notch and Delta are presented by limiting membrane and intravesicular membrane respectively and result in ligand-dependent Notch processing and activation, resembling the scenario presented for ligand-dependent Notch signaling activation in Sara endosome. The detailed regulatory mechanisms underlying Notch overactivation in retromer mutants warrants future investigation (Li, 2018).

The ability of vps35 mutant neoplastic neuroblasts to metastasize upon transplantation is intriguing. Metastasis of brain tumor cells derived from neuroblast lineages has never been observed in the developing fly larval brains, likely because the limited time window of fly larval development precludes tumor progression and metastasis. Transplantation assay, however, provides the ectopic microenvironment and allows cancer progression in a much longer time scale (months, or even years upon retransplantation). Importantly, mutations that caused metastasis of fly brain tumor cells upon transplantation have also been implicated in various human cancers. Future studies on the transcriptional profiling of the distal metastatic colonies and stepwise characterization of this long-range metastatic process promise to provide fresh mechanistic insights into the enormously complex process of cancer metastasis (Li, 2018).

Drosophila chromatin assembly factor 1 p105 and p180 subunits are required for follicle cell proliferation via inhibiting Notch signaling

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. This study reports another side of CAF1 wherein the subunits CAF1-p105 and CAF1-p180 inhibit expression of Notch target genes and shows this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt), leading to Cut downregulation and inhibition of follicle cell mitosis. These studies further show Notch is functionally responsible for these phenotypes observed in CAF1-p105/p180-deficient follicle cells. Moreover, this study reveals that CAF1-p105/p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative feedback regulatory loop between Cut and Hnt underlying CAF1-p105/p180 regulation, which is crucial for follicle cell differentiation. In conclusion, these studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner (Lo, 2019).

An agent-based model of the Notch signaling pathway elucidates three levels of complexity in the determination of developmental patterning

The Notch signaling pathway is involved in cell fate decision and developmental patterning in diverse organisms. A receptor molecule, Notch (N), and a ligand molecule (in this case Delta or Dl) are the central molecules in this pathway. In early Drosophila embryos, these molecules determine neural vs. skin fates in a reproducible rosette pattern. This study has created an agent-based model (ABM) that simulates the molecular components for this signaling pathway as agents acting within a spatial representation of a cell. The model captures the changing levels of these components, their transition from one state to another, and their movement from the nucleus to the cell membrane and back to the nucleus again. The model introduces stochastic variation into the system using a random generator within the Netlogo programming environment. The model uses these representations to understand the biological systems at three levels: individual cell fate, the interactions between cells, and the formation of pattern across the system. Using a set of assessment tools, the current model was shown to accurately reproduce the rosette pattern of neurons and skin cells in the system over a wide set of parameters. Oscillations in the level of the N agent eventually stabilize cell fate into this pattern. The dynamic timing and the availability of the N and Dl agents in neighboring cells are central to the formation of a correct and stable pattern. A feedback loop to the production of both components is necessary for a correct and stable pattern. The signaling pathways within and between cells in this model interact in real time to create a spatially correct field of neurons and skin cells. This model predicts that cells with high N and low Dl drive the formation of the pattern. This model also be used to elucidate general rules of biological self-patterning and decision-making (Reynolds, 2019).

The evolution of transcriptional repressors in the Notch signaling pathway: a computational analysis

The Notch signaling pathway governs the specification of different cell types in flies, nematodes and vertebrates alike. Principal components of the pathway that activate Notch target genes are highly conserved throughout the animal kingdom. Despite the impact on development and disease, repression mechanisms are less well studied. Repressors are known from arthropods and vertebrates that differ strikingly by mode of action: whereas Drosophila Hairless assembles repressor complexes with CSL transcription factors, competition between activator and repressors occurs in vertebrates (for example SHARP/MINT and KyoT2). This divergence raises questions on the evolution: Are there common ancestors throughout the animal kingdom. Available genome databases representing all animal clades were searched for homologues of Hairless, SHARP and KyoT2. The most distant species with convincing Hairless orthologs belong to Myriapoda, indicating its emergence after the Mandibulata-Chelicarata radiation about 500 million years ago. SHARP shares motifs with SPEN and SPENITO proteins, present throughout the animal kingdom. The CSL interacting domain of SHARP, however, is specific to vertebrates separated by roughly 600 million years of evolution. KyoT2 bears a C-terminal CSL interaction domain (CID), present only in placental mammals but highly diverged already in marsupials, suggesting introduction roughly 100 million years ago. Based on the LIM-domains that characterize KyoT2, homologues can be found in Drosophila melanogaster (Limpet) and Hydra vulgaris (Prickle 3 like). These lack the CID of KyoT2, however, contain a PET and additional LIM domains. Conservation of intron/exon boundaries underscores the phylogenetic relationship between KyoT2, Limpet and Prickle. Most strikingly, Limpet and Prickle proteins carry a tetra-peptide motif resembling that of several CSL interactors. Overall, KyoT2 may have evolved from prickle and Limpet to a Notch repressor in mammals. It is concluded that Notch repressors appear to be specific to either chordates or arthropods (Maier, 2019).

Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23

The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. This study analyzed the role of Awd in controlling Notch signaling during development. To study the awd gene function, genetic mosaic approaches were used to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. The results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments it was shown that Notch receptor accumulates in early endosome in awd mutant follicle cells. Trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. This report has demonstrated that awd is essential for Notch signaling via its endocytic role. In addition this study has identified the endocytic step at which Awd function is required for Notch signaling and evidence was obtained indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family (Ignesti, 2014).

This report demonstrates a role of awd in regulating Notch signaling via its endocytic function including surface internalization and vesicle trafficking. This conclusion is based on results that show: (1) multiple Notch target genes are mis-expressed in follicle cells and wing discs, (2) Notch accumulates in enlarged early endosomes, and (3) awd function is required for the Rab5 activity in early endosome maturation. The results also indicate that during vesicles trafficking, the Awd action is downstream of the S2 cleavage, since over-expression of NEXT (Notch EXternal Truncation) accumulated intracellularly and could not rescue the awd defect. The same NEXT over-expression strategy could rescue the shi/dynamin defect, strongly supporting the notion that the Awd action on Notch signaling is post-membrane invagination. Since over-expression of NICD could rescue the awd defect, the Awd action is likely upstream or in parallel to the S3 cleavage event (γ-secretase activity). Although a role of awd in promoting the activity of γ-secretase cannot be completely ruled out, this possibility is considered unlikely. First, awd is a known endocytic factor demonstrated in multiple tissues including neurons, trachea, and follicle cells. Second, neither the expression level nor the expression pattern of Presenilin, the catalytic subunit of γ-secretase, is altered in awd mutant cells. Third, if the defect is in γ-secretase function, it would be expected that Notch should accumulate in Hrs-positive MVBs. On the contrary, such ectopic accumulation of Notch was not observed in Hrs-positive vesicles. Therefore, the results, in aggregate, suggest that the main action of Awd on Notch signaling is via its endocytic activity promoting the transition from early endosomes to late endosomes. However, potential defects downstream of γ-secretase cleavage, such as trafficking to nucleus, in awd mutant cannot be formally ruled out (Ignesti, 2014).

One curious exception for the awd function in relation to Notch signaling is found in the border cells. During the migration of these cells, Awd expression is down-regulated. Re-expression of Awd can lead to reduction of surface receptors, such as PVR that is critical for directional movement, resulting in defective migration. Interestingly, Notch signaling is also important for border cell migration. It therefore appears that Notch signaling in these specialized cells does not require Awd activity or is insensitive to Awd protein levels. To test this, Notch expression in border cells was compared with or without Awd re-expression. In wild-type border cells (no Awd), Notch is located on the cell surface as well as in the cell body, consistent with active signaling. Forced re-expression of Awd in the border cells does not alter this pattern. This may be because Notch is already actively internalized; increasing the Awd level cannot further enhance such activity. Indeed, endocytosis is intrinsically highly active in border cells. Alternatively, the differential dependence of Notch on Awd activity may be a function of how Notch is activated, not how Awd functions differently in different cell types. For example, it has been shown that the Notch ligand Delta may be co-expressed with Notch in the same border cells. Recent reports have hinted that the requirement of endocytosis for Notch signaling may depend on the ligand-receptor relationship (for example, ligand-dependent or -independent, trans- or cis-activation, and so on). It is therefore considered that the apparent Awd-independent Notch signaling in border cells has more to do with the intrinsic Notch signaling mechanism in these cells, and less to do with the function of Awd (Ignesti, 2014).

The results indicate that the Notch signaling defect in awd mutant cells is the failure to deliver Notch past the Rab5-dependent early endosomal stage. On the other hand, the ESCRT complex mutants, which are defective in late endosome formation, promote Notch signaling. Taken together, it appears that Notch activation occurs in the intermediate stage between early endosome formation and late endosome entry. Transition from early endosomes to late endosomes is accompanied by cargo sorting, intravesicular invagination and acidification of the luminal contents. Curiously, the matured early endosome and MVB marker hrs mutant has no effect on Notch signaling, which indicates that endosomal cargo sorting per se is not required for Notch signaling. It was also shown that awd mutant cells do not exhibit altered levels of Lysotracker staining and that endosomal Notch remains on the surface of enlarged endosomes in awd mutants. The exact nature of this transition state that favors Notch processing, therefore, requires further analysis. The endocytic function of awd has traditionally been described as a 'GTP supplier' for Dynamin, based on genetic interaction data and logical extrapolation because of the GTP producing activity of Awd. This report demonstrates that, in relation to Notch signaling, awd functions downstream of, but not directly on, dynamin. It is instead critical for Rab5 activity. This is supported by the following evidence: 1) Notch in awd mutant accumulates in Avl-containing vesicles. Therefore, the awd defect is post Dynamin-mediated cleavage of membrane invagination. 2) Rab5CA can push Notch into enlarged early endosomes but failed to rescue the awd phenotype, thereby strengthening the notion that awd defect is post Shi/Dynamin function. 3) The Notch accumulation pattern in shi mutant is different from that in awd mutant. 4) Over-expression of NEXT could not rescue awd defect. The same NEXT over-expression strategy could rescue the shi defect, strongly supporting the notion that the Awd action concerning Notch signaling is post-membrane invagination. It should be noted that surface accumulation was observed of NECD antibody-detected Notch molecules, likely representing the full-length Notch not engaged in ligand binding and signaling. This indicates that Awd can affect constitutive internalization of full-length Notch (Ignesti, 2014).

The requirement of endocytosis in the signal-receiving cells for Notch activation has been amply demonstrate. It has been shown that Notch signaling in follicle cells after stage 6 requires Delta. Since this report showed that Notch signaling cannot occur in the follicle cell without awd function, it is concluded that, at least in follicle cells, endocytosis is a requisite process for ligand-dependent Notch signaling (Ignesti, 2014).

The involvement of endocytosis in Notch signaling is significant since many of the endocytic components shown to regulate Notch signaling have also been implicated in carcinogenesis. For example, V-ATPase is required for Notch signaling while mutations in ESCRT components, such as Tsg101, result in increased Notch signaling. V-ATPase has generally been considered an oncogene because it is associated with acidification of tumor cells. ESCRT components, on the other hand, have been shown to suppress tumor formation because they down-regulate surface growth factor receptor signaling. As such, attempts to design therapeutics based on these prevalent functions should take into account the effects on Notch signaling, since the relationship between Notch signaling and carcinogenesis is context-dependent (Ignesti, 2014).

Awd belongs to the Nm23 family of protein that is evolutionarily conserved from Drosophila to mammals. This in vivo analyses has demonstrated that loss of awd gene function blocks Notch signaling by altering the receptor processing after the S2 cleavage and causes Notch accumulation in early endosomes. Furthermore, evidence was obtained indicating that Awd is required for Rab5 function in early endosome formation (Ignesti, 2014).

Nm23 has been an enigmatic gene function. It is a housekeeping gene involved in nucleotide synthesis and energy metabolism, and yet exhibiting specific developmental functions. It was the first metastasis suppressor gene identified, yet exhibits oncogenic functions in some cancer cohorts. Previous work has shown that either loss-of-function or over-expression of awd can affect different aspects of epithelial morphogenesis. That is, loss-of-function awd results in over-accumulation of adherens junction components and piling up of the epithelium, while over-expression of awd results in reduced adherens junctions and disintegration of epithelial structure. These findings provided some explanation of the biphasic function of Nm23 in tumorigenesis. In light of the current studies, an additional level of complexity should be considered since Notch signaling can exert different cellular functions in different tissues and at different times during pathophysiological alterations of the same tissues (Ignesti, 2014).

Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity

Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquinated cell surface proteins. CHMP2B(Intron5) , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. This study has misexpressed CHMP2B(Intron5) in the developing Drosophila external sensory (ES) organ lineage; it was shown to be capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2B(Intron5) in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2B(Intron5) can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function (Wilson, 2019).

Activation of Arp2/3 by WASp is essential for the endocytosis of Delta only during cytokinesis in Drosophila

The actin nucleator Arp2/3 generates pushing forces in response to signals integrated by SCAR and WASp. In Drosophila, the activation of Arp2/3 by WASp is specifically required for Notch signaling following asymmetric cell division. How WASp and Arp2/3 regulate Notch activity and why receptor activation requires WASp and Arp2/3 only in the context of intra-lineage fate decisions are unclear. This study found that WASp, but not SCAR, is required for Notch activation soon after division of the sensory organ precursor cell. Conversely, SCAR, but not WASp, is required to expand the cell-cell contact between the two SOP daughters. Thus, these two activities of Arp2/3 can be uncoupled. Using a time-resolved endocytosis assay, it was shown that WASp and Arp2/3 are required for the endocytosis of Dl only during cytokinesis. It is proposed that WASp-Arp2/3 provides an extra pushing force that is specifically required for the efficient endocytosis of Dl during cytokinesis (Trylinski, 2019).

In animal cells, a thin cortex of actin filaments is dynamically regulated to produce the force required for basic cellular processes, such as motility, cytokinesis, and endocytosis. This regulation involves the nucleation of branched actin filaments by the actin-related proteins 2/3 (Arp2/3) complex (Goley, 2006, Pollard, 2007, Rotty, 2013). By itself, Arp2/3 is weakly active, and nucleation-promoting factors (NPFs) are needed to stimulate its nucleation activity. Thus, when and where actin-based pushing forces are produced in the cell depends on the localization and activity of the NPFs. Wiskott-Aldrich syndrome protein (WASP) family proteins are the best-studied NPFs. These are usually maintained in an autoinhibited state and can be activated at the membrane by small GTPases. Three WASP family members are known in Drosophila: WASp, SCAR/WAVE (suppressor of cyclic AMP repressor/WASp-family verpolin-homologous protein), and WASH (WASp and SCAR homolog). Genetic analysis indicates that SCAR is the primary NPF in Drosophila, since the loss of SCAR activity leads to developmental and cellular defects that are similar to those seen upon the disruption of Arp2/3 activity, whereas WASH has a non-essential function during oogenesis, and WASp is only required for specific Notch-mediated fate decisions following asymmetric cell divisions in muscle, brain, and sensory organ lineages. This function of WASp is mediated by Arp2/3, since the loss of the Arp3 and Arpc1 subunits of the Arp2/3 complex leads to WASp-like cell fate defects. How WASp and Arp2/3 regulate Notch signaling is unclear. In addition, given the ubiquitous expression of WASp and the functional pleiotropy of Notch, it is unclear why WASp is only required for Notch signaling in the context of asymmetric cell division (Trylinski, 2019).

Notch receptor activation requires a pulling force to expose an otherwise buried cleavage site in the extracellular domain of Notch, the cleavage of which eventually produces the Notch intracellular domain (NICD). Previous studies have shown that endocytosis of the Notch ligands provides a strong enough pulling force to direct receptor activation. Since WASp and Arp2/3 are known to increase the efficiency of endocytosis by nucleating branched filaments shortly after membrane ingression begins (i.e., when high forces are required), it is conceivable that WASp-stimulated Arp2/3 activity may facilitate receptor activation by regulating the endocytosis of the Notch ligand Delta (Dl). However, it was reported that the endocytosis did not depend on Arp3, clearly arguing against this model. It was proposed that Arp2/3 may instead regulate the transport of endocytosed Dl back to the apical membrane, where it would activate Notch. This model, however, is not supported by a recent photo-tracking analysis of fluorescent Notch receptors, showing that signaling takes place along the lateral membrane following asymmetric division. NICD was produced during cytokinesis from a subset of Notch receptors that are located basal to the midbody (Trylinski, 2017). Thus, how WASp-Arp2/3 positively regulates Notch signaling is not known (Trylinski, 2019).

Early loss of Notch signaling in WASp and Arp3 mutants did not merely result from a defect in pIIa-pIIb contact expansion at cytokinesis. Contact expansion involves the activation of Arp2/3 by Rac and SCAR, but not by WASp, and SCAR and Rac are dispensable for Notch activation during cytokinesis and pIIa specification. Thus, Arp2/3 has separable functions in contact expansion and Notch signaling at cytokinesis. Instead, this detailed analysis of the endocytosis of Dl revealed that WASp is required for the efficient endocytosis of Dl during cytokinesis, but not afterward. This specific requirement of WASp and Arp2/3 for endocytosis during cytokinesis only may explain its specific requirement in Notch-mediated intra-lineage decision (Trylinski, 2019).

This study showed that Arp2/3 has two separable activities in asymmetric cell divisions: Arp2/3 promotes the rapid expansion of the new cell-cell contact and stimulates the endocytosis of Dl from this cell-cell contact to regulate intra-lineage fate decisions by Notch. These two activities involve distinct NPFs. SCAR, downstream of Rac, promotes the formation of a dense F-actin network around the midbody to generate a force that regulates cell-cell contact between sister cells and facilitates withdrawal of the membranes of the neighboring cells. SCAR, however, is largely dispensable for Notch receptor activation, suggesting that the force required for contact expansion is not key for Notch receptor activation. In contrast, WASp is required for Notch signaling but is dispensable for contact expansion during cytokinesis. While these two functions of Arp2/3 are separable, a functional interplay is possible, if not likely. For instance, Rac and SCAR may facilitate the activity of WASp in Dl endocytosis through the recruitment of Arp2/3 along the pIIa-pIIb interface (Trylinski, 2019).

Before the present study, WASp-mediated activation of Arp2/3 was thought to regulate the intracellular trafficking of internalized Dl, not its endocytosis. This model assumed that Dl signals at the apical membrane, which seems unlikely since it was recently shown that NICD originates from the lateral membrane during cytokinesis (Trylinski, 2017). Using a time-resolved endocytosis assay, this study showed that WASp-mediated activation of Arp2/3 is required to promote the endocytosis of Dl during cytokinesis, but not afterward. The specific time window during which the activities of WASp and Arp3 are required may explain why this requirement had previously been missed. In analogy to the role of WASp in yeast, it is proposed that WASp is recruited at sites of Dl endocytosis to form a branched actin network that provides an inward pushing force onto the invaginated membrane. This would increase the efficiency of endocytosis-hence the rate of the force-dependent activation of Notch. Accordingly, WASp would play a modulatory role, which is critical within a defined time window. Consistent with this view, the WASp mutant bristle phenotype can be suppressed by lowering the threshold for NICD levels in flies with reduced levels of the CSL co-repressor Hairless (Trylinski, 2019).

Why are WASp and Arp3 required only during cytokinesis for the endocytosis of Dl? WASp is likely to have a general function in endocytosis in Drosophila, as in other organisms, and may therefore regulate the endocytosis of many cargoes, including Dl, throughout development. Consistent with a general function of WASp, it is ubiquitously expressed and is not specifically upregulated in sensory lineages. However, the role of WASp-activated Arp2/3 in endocytosis is essential, at the organismal level, only in the context of intra-lineage decisions regulated by Notch. This specificity may be explained by when Notch signals, namely, at the end of mitosis. It is well established that clathrin-mediated endocytosis is shut down at mitosis and is progressively restored during cytokinesis. One mechanism contributing to this inhibition throughout mitosis is increased membrane tension. Since an increased requirement for actin is observed in cells in which membrane tension is high, it is speculated that the endocytosis of Dl more critically depends upon actin regulation by WASp during cytokinesis due to increased membrane tension. In other words, it is proposed that the pushing force provided by WASp-induced F-actin is needed for the efficient endocytosis of Dl to counteract the increased membrane tension associated with mitosis. Thus, while WASp and Arp2/3 likely play a general role in endocytosis, their activities become critical for the mechanical activation of Notch only when the inhibition of endocytosis needs to be overcome in late mitosis (Trylinski, 2019).

The requirement of WASp for Notch receptor activation is symmetric to those of Epsin, a conserved endocytic adaptor that helps generate the force for membrane invagination during endocytosis. Epsin is generally required for ligand endocytosis and Notch signaling in flies and mammals, with the exception of Notch-mediated intra-lineage decisions, as revealed by the development of sensory bristles in epsin mutant clones. It is speculated that the inhibition of Epsin at mitosis, possibly via its phosphorylation by CDK1/Cdc2, renders necessary the extra pushing force provided by WASp for the efficient endocytosis of Dl (Trylinski, 2019).

In summary, a model is proposed whereby the activity of WASp-Arp2/3 generally increases the efficiency of endocytosis and becomes specifically required only during cytokinesis, when Dl activates Notch to mediate intra-lineage decisions. This model may be general and apply to mammalian tissues where Notch is known to regulate intra-lineage decisions. N-WASp, the ubiquitously expressed WASp in mammals, is required for the maintenance of skin progenitor cells and hair follicle cycling in the mouse, and Notch plays a critical role in the self-renewal of skin stem cells. Whether Notch signaling is regulated by N-WASp in this context remains to be examined (Trylinski, 2019).

list of proneural and neurogenic genes


Bossing, T., et al. (1996). The embryonic central nervous system lineages of Drosophila melanogaster. I. Neuroblast lineages derived from the ventral half of the neuroectoderm. Dev. Biol 179: 41-64. 8873753

Cummings, C.A. and Cronmiller, C. (1994). The daughterless gene functions together with Notch and Delta in the control of ovarian follicle development in Drosophila. Development 120: 381-94. 8149916

Da Ros, V. G., Gutierrez-Perez, I., Ferres-Marco, D. and Dominguez, M. (2013). Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis. PLoS Biol 11(5): e1001554. PubMed ID: 23667323

Farnsworth, D. R., Bayraktar, O. A. and Doe, C. Q. (2015). Aging neural progenitors lose competence to respond to mitogenic Notch signaling. Curr Biol 25: 3058-3068. PubMed ID: 26585279

Ghysen, A. and Dambly-Chaudiere, C. (1988). From DNA to form: The achaete-scute complex. Genes Dev. 2: 495-501

Goley, E. D. and Welch, M. D. (2006). The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 7(10): 713-726. PubMed ID: 16990851

Gomez-Skarmeta, J. L., et al. (1995). Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs. Genes Dev. 9: 1869-1882. 7649474

Gonzalez-Gaitan, M. and Jackle, H. (1995). Invagination centers within the Drosophila stomatogastric nervous system anlage are positioned by Notch-mediated signaling which is spatially controlled through wingless. Development 121: 2313-25. 7671798

Hartenstein, A.Y., Rugendorff, A., Tepass, U. and Hartenstein, V. (1992). The function of the neurogenic genes during epithelial development in the Drosophila embryo. Development 116(4): 1203-1220. 1295737

Ignesti, M., Barraco, M., Nallamothu, G., Woolworth, J. A., Duchi, S., Gargiulo, G., Cavaliere, V. and Hsu, T. (2014). Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23. BMC Biol 12: 12. PubMed ID: 24528630

Lo, P. K., Huang, Y. C., Corcoran, D., Jiao, R. and Deng, W. M. (2019). Drosophila chromatin assembly factor 1 p105 and p180 subunits are required for follicle cell proliferation via inhibiting Notch signaling. J Cell Sci. PubMed ID: 30630896

Li, B., Wong, C., Gao, S. M., Zhang, R., Sun, R., Li, Y. and Song, Y. (2018). The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking. Elife 7. PubMed ID: 30176986

Maier, D. (2019). The evolution of transcriptional repressors in the Notch signaling pathway: a computational analysis. Hereditas 156: 5. PubMed ID: 30679936

Panchal, T., Chen, X., Alchits, E., Oh, Y., Poon, J., Kouptsova, J., Laski, F. A. and Godt, D. (2017). Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam. PLoS Genet 13(5): e1006790. PubMed ID: 28542174

Pollard, T. D. (2007). Regulation of actin filament assembly by Arp2/3 complex and formins. Annu Rev Biophys Biomol Struct 36: 451-477. PubMed ID: 17477841

Reynolds, E. R., Himmelwright, R., Sanginiti, C. and Pfaffmann, J. O. (2019). An agent-based model of the Notch signaling pathway elucidates three levels of complexity in the determination of developmental patterning. BMC Syst Biol 13(1): 7. PubMed ID: 30642357

Rotty, J. D., Wu, C. and Bear, J. E. (2013). New insights into the regulation and cellular functions of the ARP2/3 complex. Nat Rev Mol Cell Biol 14(1): 7-12. PubMed ID: 23212475

Schmidt, H., et al. (1997). The embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neuroectoderm. Dev. Biol. 189(2): 186-204

Schweisguth, F. (1995). Suppressor of Hairless is required for signal reception during lateral inibition in the Drosophila pupal notum. Development 121: 1875-1884. 7601001

Skeath, J. B. et al. (1992). Gene regulation in two dimensions: the proneural achaete and scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Genes Dev 6: 2606-19. 7601001

Skeath, J. B., Panganiban, J. F. and Carroll, S. B. (1994). The ventral nervous system defective gene controls proneural gene expression at two distinct steps during neuroblast formation in Drosophila. Development 120: 1517-1524. 8050360

Trylinski, M., Mazouni, K. and Schweisguth, F. (2017). Intra-lineage fate decisions involve activation of Notch receptors basal to the midbody in Drosophila sensory organ precursor Cells. Curr Biol 27(15): 2239-2247 e2233. PubMed ID: 28736165

Trylinski, M. and Schweisguth, F. (2019). Activation of Arp2/3 by WASp is essential for the endocytosis of Delta only during cytokinesis in Drosophila. Cell Rep 28(1): 1-10. PubMed ID: 31269431

Wilson, C., Kavaler, J. and Ahmad, S. T. (2019). Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity. Dev Neurobiol. PubMed ID: 31587468

Wu, Y. C., Lee, K. S., Song, Y., Gehrke, S. and Lu, B. (2017). The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain. PLoS Genet 13(5): e1006785. PubMed ID: 28520736

Yatsenko, A. S. and Shcherbata, H. R. (2018). Stereotypical architecture of the stem cell niche is spatiotemporally established by miR-125-dependent coordination of Notch and steroid signaling. Development 145(3). PubMed ID: 29361571

Zhou, W., He, Q., Zhang, C., He, X., Cui, Z., Liu, F. and Li, W. (2016). BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development. Elife [Epub ahead of print]. PubMed ID: 27719760

Zhu, J., Palliyil, S., Ran, C. and Kumar, J. P. (2017). Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation. Proc Natl Acad Sci U S A 114(23): 5846-5853. PubMed ID: 28584125

Zygotically transcribed genes

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