The Interactive Fly

Zygotically transcribed genes

Proneural and Neurogenic Genes

Notch Signaling

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).

Asmar, J., Biryukova, I. and Heitzler, P. (2008). Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression. Dev. Biol. 316(2): 487-97. PubMed Citation: 18329012

Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression

The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). This study investigated, through new genetic tools, the function of dLMO, the Drosophila LIM only transcription factor that was already known to control wing development. dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression (Asmar, 2008).

The lack of dLMO protein leads to very distinctive phenotypes. The mutant animals are not able to fly, they have a short life span and show an abnormal gait behaviour. In addition, they show a discreet bristle phenotype. In Drosophila, there are two paralogous LMO factors, dLMO and CG5708. These genes are expressed in the CNS where redundancy is not excluded. However CG5708 is not expressed in the wing discs and presumptive SOPs. Therefore it is concluded that the mild phenotype observed for the adult PNS in dLMO mutants, is not attributed to gene redundancy. dLMO encodes two distinct isoforms, dLMO-PA and -PB, which only differ from their N-terminus. Only dLMO-RA is broadly expressed in the notum, and contributes to the PNS phenotype. dLMO function is also critical in the developing central nervous system for the activity of the ventral lateral neurons, LNvs. It is highly probable that dLMO-RB has some subtle biological activities in the brain, where it has a specific pattern (Asmar, 2008).

In vertebrate, multiproteic complexes composed by GATA-1, LMO2, Ldb-1 and the bHLHs E47 and SCL, are required for normal differentiation of haematopoietic cells. The current results highlight several evidences in favour of dLMO as a GATA coactivator in Drosophila . (1) A genetic synergism exists between pnr and dLMO null alleles. (2) dLMO modulates the activity of a DC:ac-lacZ reporter, the model target of Pnr, in vivo. Loss of function dLMO mutants show reduced level of the DC:ac-lacZ expression, whereas in gain-of-function dLMO mutants the DC:ac-lacZ expression is increased. (3) dLMO-PA isoform directly interact with Pnr in GST pull down assay. Therefore it is concluded that dLMO might enhance the proneural activity of Pnr through direct interaction with the GATA factor. Consistently, dLMO expression overlaps with the dorsal-most domain of Pnr during third instar larval stages. Though Pnr controls the development of both DC and SC bristles, dLMO null alleles affect only DC bristles. dLMO expression, that overlaps both SC and DC proneural clusters in the notum, is significantly weaker in the SC region, suggesting that regulation of proneural ac/sc expression is differentially sensitive and responds to local combinations of transcription factors. These data support previously published studies demonstrating that the proneural activity of Pnr is prominently repressed in the SC region by the LIM-HD transcription factor Isl (Asmar, 2008).

At later stages, dLMO expression is excluded from the corresponding SOP and its derivative cells. In contrast, the proneural factor Sens, that plays an important role for sensory organ specification, is first broadly expressed in proneural clusters at low levels where it functions as a repressor of ac/sc, and then later, is expressed at high levels in the presumptive SOPs, where it acts as a transcriptional activator that directly interacts and synergizes with the proneural proteins, Ac and Sc. It has been shown that both Gfi-1 and GATA-1, the mammalian ortholog of Sens and Pnr respectively, are essential for development of the closed related erythroid and megakaryocytic lineages. The Sens/Pnr interaction is evolutionary conserved in Drosophila neurogenesis. It is suggestd that Pnr could participate to the Sens-dependent positive autoregulation of Ac/Sc in late SOPs where dLMO is not expressed. The synergism between Pnr and Sens would need more detailed investigations. Taken together, these studies have shown dLMO-PA as a co-activator for Pnr during the establishment of proneural fields and revealed another level of proneural ac/sc regulation during late neurogenesis in the Drosophila PNS (Asmar, 2008).

Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin: Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages

The basic helix-loop-helix (bHLH) proneural proteins Achaete and Scute cooperate with the class I bHLH protein Daughterless to specify the precursors of most sensory bristles in Drosophila. However, the mechanosensory bristles at the Drosophila wing margin have been reported to be unaffected by mutations that remove Achaete and Scute function. Indeed, the proneural gene(s) for these organs is not known. This study shows that the zinc-finger transcription factor Senseless, together with Daughterless, plays the proneural role for the wing margin mechanosensory precursors, whereas Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages. Evidence is provided that Senseless and Daughterless physically interact and synergize in vivo and in transcription assays. Gain-of-function studies indicate that Senseless and Daughterless are sufficient to generate thoracic sensory organs (SOs) in the absence of achaete-scute gene complex function. However, analysis of senseless loss-of-function clones in the thorax implicates Senseless not in the primary SO precursor (pI) selection, but in the specification of pI progeny. Therefore, although Senseless and bHLH proneural proteins are employed during the development of all Drosophila bristles, they play fundamentally different roles in different subtypes of these organs. The data indicate that transcription factors other than bHLH proteins can also perform the proneural function in the Drosophila peripheral nervous system (Jafar-Nejad, 2006).

In 1978, García-Bellido and Santamaria reported that ac and sc are required for the generation of the majority of the Drosophila bristles. The large body of work that followed this discovery led to the realization that Ac and Sc are members of the bHLH proneural protein family, which are involved in early steps of neurogenesis in flies and vertebrates. Later, two other bHLH genes, atonal and amos, were shown to play the proneural role for almost all SOs that did not depend on Ac and Sc function, with the notable exception of the wing margin (WM) mechanosensory bristles (Garcia-Bellido, 1978). This study shows, based on multiple lines of evidence, that Sens plays the proneural role for these bristles: sens expression in the WM begins before the selection of mechanosensory pIs in a proneural cluster, similar to other proneural proteins; sens expression is upregulated in presumptive pIs and is downregulated in ectodermal cells, just like ac and sc expression is refined to pIs in thoracic proneural clusters; loss and gain of sens function result in loss and gain of SOs in the wing; and Sens synergizes with the Da protein in vivo and in transcription assays, and binds Da in a GST pull-down assay. Unexpectedly, overexpression of the anti-apoptotic protein P35 in the WM results in the generation of a large number of neurons along the PWM, uncovering the neural identity of the PWM bristle precursors. Similar to the AWM (anterior wing margin), the expression pattern and loss of-function phenotype of sens in the PWM (posterior wing margin) indicate a proneural role for sens for the PWM bristles as well. However, the neural potential of the PWM bristles is not realized in the wild-type situation because of apoptosis of the pI progeny, providing an example of the role of apoptotic machinery in diversifying the various sensory lineages. In summary, Sens satisfies all the genetic and developmental criteria for being a proneural protein for the WM bristles, and is the only zinc finger protein shown to play a proneural role in SO development in flies (Jafar-Nejad, 2006).

As for other proneural proteins, the proneural function of Sens requires the function of Da. Da serves as the binding partner for the bHLH proneural proteins to bind E-box sequences and is also able to bind DNA as homodimers. No function has been assigned to Da homodimers in Drosophila, largely because of the identification of tissue-specific bHLH proteins in most contexts in which Da functions. In the WM mechanosensory precursors, however, none of the known tissue-specific bHLH proneural proteins is expressed, suggesting a proneural role for Da homodimers. One might argue that there is probably an unknown dimerization partner for Da in these sensory precursors, and this possibility cannot be excluded. However, two groups have independently identified all Drosophila genes encoding bHLH proteins using database searches of the complete Drosophila genome and none of the newly identified bHLH proteins are predicted to be a transcriptional activator of the Ac-Sc or Atonal families. Also, none of these genes shows an embryonic expression pattern compatible with a proneural function for the CNS. Because da is required for mechanosensory organ formation, and as it can efficiently generate bristles in the absence of ASC, it is proposed that Da homodimers cooperate with Sens to endow neural identity to AWM mechanosensory organs and PWM bristle precursors. The physical interaction of these two proteins and the strong transcriptional synergy between them strongly favors a role in activating key target genes in SO development (Jafar-Nejad, 2006).

These data also reveal that Ac and Sc promote the survival of the WM mechanosensory neurons and support cells independently of pI selection. The more severe loss of neurons compared with support cells associated with the loss of Ac and Sc in sc10-1 suggests either that the neurons (or their precursors) are more sensitive to the lack of ac and sc function, or that the loss of support cells is secondary to the neuronal death. The observation that adding or removing one copy of wild-type sens strongly modifies the sensory lineage apoptosis observed in sc10-1 animals indicates that, in addition to a proneural function, Sens also plays an anti-apoptotic role in these cells; this is in agreement with many reports on the role of sens and its homologues in mammals and C. elegans in preventing apoptosis. It is interesting to note that although Ac and Sc are not detected in the PWM by antibody staining, P35 overexpression rescues many more neurons in the PWM of wildtype flies than in sc10-1 animals. This indicates a requirement for Ac and Sc in these cells (Jafar-Nejad, 2006).

During the third instar larval period, low levels of Sens are expressed in the proneural clusters along the AWM that will give rise to the pI cells of the AWM chemosensory bristles. Using in vivo and in vitro assays, it has been shown that low levels of Sens repress, and high levels of Sens activate, ac and sc expression in these proneural clusters, and thereby that Sens is involved in pI selection. Given the similar low-level expression of Sens in thoracic microchaetae proneural clusters and the severe loss of microchaetae in adult sens clones, it had been hypothesized that Sens also functions during proneural upregulation and in the selection of the microchaetae pIs. It was therefore surprising to find that microchaetae pI selection does not require Sens function. Data has been presented on the function of the adaptor protein Phyllopod and its relationship with Sens in microchaetae development. Sens was shown to be required for the function of Phyllopod in the pIs, as well as for timely downregulation of phyllopod expression in epidermal cells. This suggests a dual role for Sens in pIs and surrounding epidermal cells, in agreement with the binary switch model. In contrast, phyllopod expression can still be upregulated in single cells in sens mutant clones, suggesting that pI selection is not disrupted. This study now presents evidence that microchaetae pIs are indeed selected in sens clones and that they divide to generate progeny. However, the mutant pIs exhibit an abnormal division pattern, and a pIIa-to-pIIb transformation is observed, as evidenced by a gain of neurons at the expense of support cells. These data indicate that Sens regulates several aspects of microchaetae precursor development after the pIs are selected (Jafar-Nejad, 2006).

In summary, the normal development of all adult bristles in flies relies on the function of Ac and Sc, Da and Sens. The data indicate that despite the structural and functional similarities between various adult bristles, sens functions at four distinct steps in different lineages. First, in the WM mechanoreceptor and noninnervated lineages, very high levels of Wingless induce the expression of Sens, which assumes a true proneural role and specifies SO fate independently of the typical proneural proteins Ac and Sc. Second, in the WM chemosensory lineages, for which ac and sc are the proneural genes, Sens is required for pI selection, as evidenced by the observation that it represses proneural gene expression in ectodermal cells and activates proneural gene expression in presumptive pIs. Third, even though gain-of-function studies show that Sens is able to induce pI formation in the thorax in the absence of Ac and Sc function, it normally plays a later role in specification of the pIIa versus the pIIb of microchaetae lineages. Fourth, Sens is required for the survival of the pI progeny in the WM mechanosensory lineages. It was also found that ac and sc prevent apoptosis in this lineage independently of pI specification. Finally, the data suggest that a typical Da heterodimeric complex is not required during the formation of the WM mechanosensory and noninnervated bristle pIs. Hence, the cooperation between the same group of genes is adapted in different ways to ensure the proper development of various SOs (Jafar-Nejad, 2006).

The Sens homolog Gfi1 plays important roles in several developmental processes, including inner ear hair cell development, hematopoietic stem cell self-renewal rate, intestinal cell fate specification and neutrophil differentiation. Moreover, Gfi1 has an oncogenic potential and has been implicated in several human diseases, such as hereditary neutropenia, spinocerebellar ataxia type 1 and small cell lung carcinoma. Therefore, given the structural and functional similarities between Gfi1 and Sens, further analysis of the various aspects of Sens function in Drosophila SO development will continue to help unravel the mechanisms of Gfi1 function in health and disease (Jafar-Nejad, 2006).

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).

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, this study monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). The scrib mutation was shown to impact the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally this work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth (Logeay, 2022).

In this study, using Notch-driven paradigms of epithelial overgrowth in Drosophila wing discs, the molecular mechanisms are described underlying the cooperation between Notch and polarity loss during neoplasia. It was shown that epithelial polarity alterations redirect the transcriptional outcome of the Notch signaling pathway, thus defining a specific set of new neoplastic Notch direct targets. It was further shown that this redirection occurs mainly on pre-existing Su(H)-bound regions rather than new ones. Finally, it was shown that, similar to what was previously described for Ras signaling, the AP-1/Stat/Yki/Ftz-f1 transcription factors are required for the cooperation between Notch signaling and polarity loss during neoplastic growth (Logeay, 2022).

Although cancer genomes exhibit multiple mutations in cancer cells, their functional interactions remain difficult to monitor and model. Neoplastic tissues, generated upon the combination of Notch pathway activation and polarity loss through scrib mutation, experience many cellular stresses: DNA-damage responses, but also endoplasmic reticulum and unfolded protein response, starvation or oxidative stresses. However, even though present, these different stresses, in particular oxidative stress and DNA damage, are not individually necessary in the context of polarity loss as blocking them or the cellular response they promote (by CAT/SOD overexpression, or inhibition of p53) did not significantly suppress the NS tumorous behaviors. These observations suggest that the different stress pathways activated during polarity loss are either not required for fueling growth (they are rather a consequence than a cause of neoplastic growth), or might act 'redundantly' to activate a common core response required for increased growth (Logeay, 2022).

Although Drosophila and mouse models have demonstrated that overactive signaling pathways cooperate with epithelial polarity impairment to generate neoplastic growth, the vast majority of studies seeking to understand the underlying mechanisms have focused primarily on the cooperation between activated RasV12 and scrib mutants, especially in Drosophila. Importantly, the current study, investigating the cooperation between Notch and polarity, shows that many observations made for Ras can be extended to Notch, suggesting that the paradigms used are not specific to Ras but might represent a more general tumor growth paradigm. But, because the main, if not only, Notch pathway outcome is transcriptional, the NICD/scrib- model allowed the modes of cooperation to be studied in greater detail. The cooperation between Notch pathway activation and polarity loss led to a specific transcriptional program, and in particular the activation of new Notch direct targets. This was not the consequence of a general redeployment to new target gene loci of Su(H), the Notch pathway-dedicated transcription factor, ruling out one possible model for the oncogene/polarity cooperation. Thus, what could be the mechanisms controlling which genes were activated in the different conditions? All 'Notch'-activating transcriptional complexes comprise NICD, Mastermind and Su(H). Although no differences in overall levels of NICD and Su(H) could be detected by western blot (data not shown), they could be modified in different ways post-translationally leading to different Notch responses (e.g. core Notch response, N-only, NS-only). Indeed, recent reports point towards different post-translational modifications for Su(H). However, whether they lead to different transcriptional programs, and whether they occur in vivo in the N and NS models, remain to be studied. Through the use of iRegulon, we demonstrated that the genes of the NS transcriptome, and most importantly the NS Notch direct targets, were enriched in their regulatory regions for elements corresponding to specific transcription factors, and in particular Stat92E or bZIP factors. The fact that similar transcription factor families were found in the overall group of upregulated genes and in the more limited subset of Notch direct genes suggests that the Notch output was controlled, at least in part, by factors that act more broadly on the genome. These analyses support a model in which polarity loss redirects the output of the Notch transcriptional program by the action of cooperating transcription factors. However, further work, such as detailed comparative ChIP analyses of the different factors in the different conditions, is required to establish this model firmly (Logeay, 2022).

Although this study demonstrated the involvement of a similar 'oncogenic module' as identified for the RasV12/scrib- neoplastic model, there are specifics that are likely oncogene specific. First, unlike what was reported for RasV12/scrib- transcriptomes, Yki/Sd/TEAD modules were not found to be enriched in the different Notch and scrib- transcriptomes. In the case of Ras, it has been shown that Yki activity can reprogram Ras by promoting the expression of the Ras pathway-specific regulators Capicua and Pointed to promote aggressive growth. Both genes were either unaffected (capicua) or downregulated (pointed) in the NS Notch-driven neoplastic paradigm, suggesting that, even though Yki is clearly required, changes in the expression of capicua and pointed are unlikely to be mediators here. These differing results in the enrichment of Yki/Sd/TEAD motifs between Notch and Ras transcriptomes in the context of polarity loss might reflect the inhibitory effect Notch has on Yki activity in the wing pouch, in part through the action of vestigial. Furthermore, in the NS transcriptome, a contribution was identified of the E(spl) bHLH transcriptional repressors, canonical Notch targets, which represents thus a Notch specificity. However, the fact that motifs for E(spl)-HLH repressors are found in the upregulated transcriptome of NS and not N could suggest that in NS the repressive ability of E(spl)-HLH factors is antagonized, further allowing higher expression of Notch targets. More precisely, the previous work identified many incoherent feed-forward loops in the N hyperplastic transcriptome, including through the action of E(spl) repressors, which might thus be resolved in NS. It would be interesting to explore further the link between NS and E(spl)-HLH-mediated repression, but due to the high redundancy between the seven E(spl)-HLH factors (Δ, &gamma', β, 3, 5, 7, 8) and Dpn, the requirement of E(spl)-HLH-mediated repression in Notch-driven neoplasia could not be formally tested (Logeay, 2022).

By performing functional assays to identify the genes and processes required for NS tumor growth, this study demonstrated that the Notch direct targets associated with 'de novo' NS-specific Su(H) peaks were unlikely to be major contributors. We did show, however, that the bZIP PAR domain-containing factor Pdp1 is required for NS tumor growth and invasiveness. Su(H) is bound in the vicinity of Pdp1 in all wing discs set-ups, and in particular in N and NS, and Pdp1 represents a 'core' Notch target activated in all overgrowth conditions, albeit at higher levels in polarity-deficient conditions. Pdp1 is not only a Notch target, but also a Jak/Stat target, at least in the developing eye, and canonical tandem Stat92E putative binding sites are found in its second intron, although not overlapping with Su(H) binding, which is found in its first intron. Interestingly, Pdp1 is required for Stat92E phosphorylation and efficient Jak/Stat signaling, suggesting that Notch might amplify Stat92E signaling during wing disc neoplastic growth, both through ligand expression (Upd ligands are Notch direct targets) and Pdp1 expression (Logeay, 2022).

Although Pdp1 downregulation could suppress NS neoplastic growth, it was not as efficient as JNK inhibition, or Yki downregulation, suggesting that other factors in parallel to Pdp1 might be involved, such as the previously identified Atf3, but also the other Notch direct target Ets21C. Indeed, RNAi-mediated knockdown of Atf3 or Ets21C partly suppressed Bx>NS tumor growth (GFP) and invasiveness (Mmp1). This action of both Pdp1 and Ets21C suggest a feed-forward loop downstream of Notch that in the context of polarity loss and JNK activity promotes neoplastic growth. However, given that Atf3, Pdp1 and Ets21C (but also Ftz-f1) are all upregulated in N hyperplastic conditions, their sole upregulation cannot be sufficient for neoplasia. The fact that Atf3 and Pdp1 iRegulon enrichments are not found in N could indicate that, despite being upregulated in hyperplastic N, their transcriptional activities are hindered, or that one key cooperating factor enabling their action is missing. Further studies are thus required to test this possibility and study how, in the context of normal epithelial polarity, Notch activation prevents the action of Pdp1/Ets21C/Atf3, thus preventing the transition to neoplasia (Logeay, 2022).

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).

Activation of the Notch signaling pathway in vivo elicits changes in CSL nuclear dynamics

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, this study developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or 'assisted' loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which is proposed to confer sensitivity to small input changes and facilitate timely shut-down (Gomez-Lamarca, 2018).

Until recently, most existing models have portrayed CSL as a molecule with long DNA residence that serves as a static platform for exchange between NICD and co-repressors. This analysis, using a combination of FRAP and single-molecule tracking (SMT) to measure Su(H) dynamics, reveals a very different story and highlights two important characteristics. First, in Notch-OFF conditions, Su(H) normally undergoes very transient DNA residency, despite the fact that it is important for repression of the target loci. This implies that prolonged binding is not a prerequisite for repression. It also argues against a model where co-factors are exchanged while CSL remains bound to DNA. Second, in Notch-ON conditions, there is a striking enrichment of Su(H) at E(spl)-C, its primary target locus, where its dwell time is significantly increased. These changes in CSL-binding dynamics, can enable a sensitive and accurate response to NICD at its target sites (Gomez-Lamarca, 2018).

This study has found that NICD enhances both Su(H) recruitment and residence time at its target locus E(spl)-C, via a combination of mechanisms. One key step is that NICD-Su(H) complexes induce local changes in chromatin, which requires Trr (MLL3/4), a long-range co-activator that can contribute to chromatin opening. Notably, the consequence of NICD-induced chromatin opening is that it renders the target enhancers more accessible for additional complexes, regardless of whether they contain NICD or Hairless. Since binding of Hairless and NICD to Su(H) are mutually exclusive, it is likely that these represent discrete activator (Su(H)-NICD) and repressor (Su(H)-Hairless) complexes, although this study has not formally shown that Hairless recruitment relies on Su(H). This enhanced recruitment by NICD resembles that described for the glucocorticoid receptor and other factors, referred to as 'assisted loading,' whereby the binding of one protein complex helps the binding of another. It is proposed that the localized chromatin remodeling brought about by Su(H)-NICD reduces obstacles (e.g., moves nucleosomes) to facilitate DNA binding, i.e., effectively increasing KON. Such indirect cooperativity would render the response very sensitive to signal levels (Gomez-Lamarca, 2018).

A second aspect helps explain how the transiently bound Su(H)-NICD complexes can successfully activate transcription. Although at genomic locations with paired binding motifs the dimerization of NICD could enhance binding, the data argue that the presence of Mam itself confers a longer dwell time to the activator complex, most likely by favoring contacts with additional chromatin-associated factors, such as Mediator complex. One candidate to mediate these effects was CBP, a histone acetyltransferase that interacts with Mam and is necessary for its ability to stimulate transcription. However, inhibiting CBP or depleting the Mediator subunit Med7 only slightly modified the Su(H) dynamics, suggesting that each makes at best a modest contribution to the change in its behavior. As neither manipulation fully replicated the effects of Mam inhibition/depletion, despite preventing transcriptional activation, it is likely that they also act at a later step in the initiation process. Thus Mam is likely to exert its early effects on Su(H) recruitment through a combination of other chromatin factors besides CBP. The interaction of the tripartite Su(H)-NICD-Mam complex with these chromatin factors, although still transient, could confer a probabilistic switch between an inactive state and an active state, by leaving a longer-lasting modification or reorganization of the chromatin template or initiation complex (Gomez-Lamarca, 2018).

The fact that the Su(H)-NICD activator complex also enhances recruitment of Hairless co-repressor complexes was entirely unexpected based on prevailing models, and has several important consequences. First, it will bring opposing enzymatic activities (e.g., both histone acetyl-transferases and histone deacetylases), which could create a covalent modification cycle with switch-like properties, potentially further sensitizing responses to Notch. Second, enhanced recruitment of Hairless would ensure that genes are rapidly turned off after the signal decays, the switch operating in the converse direction when NICD levels decrease. Such 'facilitated repression,' where transcriptional activators promote global chromatin decondensation to facilitate loading of repressors, has also been described during circadian gene regulation where it operates as an amplitude rheostat (Gomez-Lamarca, 2018).

In conclusion, in vivo analysis of the mechanisms underlying the transcriptional response to Notch signaling reveal the fundamental importance of changes in DNA-binding dynamics and highlight how different mechanisms combine to enhance Su(H) recruitment and dwell time at E(spl)-C in Notch-ON cells. Whether both mechanisms operate at all Notch-regulated loci remains to be established, but they will likely be relevant for most genes where CSL occupancy was found to increase in Notch-ON conditions. Furthermore, this new insight into Notch signaling leads to a proposal that similar changes in the dynamics of nuclear effectors may also operate to deliver proper transcriptional outputs of other key signaling pathways (Gomez-Lamarca, 2018).

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).

ATF4-induced Warburg metabolism drives over-proliferation in Drosophila

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity (Sorge, 2020).

Controlling cell proliferation is one of the major challenges of multicellular life, both during phases of growth in developing organisms and phases of homeostatic cell replenishment essential in adult animals. Lack of appropriate control can lead to severe disorders, including cancer, at any stage of life. While over-proliferation of transformed, cancerous cells is usually caused by inactivation of tumor suppressors and/or activation of oncogenes, it has long been noted that tumors exhibit altered cellular characteristics such as a glycolytic metabolism. While this metabolic switch has been shown to be caused by oncogene signaling, cellular metabolism is also controlled at multiple levels under normal physiological (non-transformed) conditions, including at the transcriptional level through diverse stress-response pathways. One of these is the activating transcription factor 4 (ATF4), which is known to activate a transcriptional (integrated) stress response (ISR) under various stress conditions that trigger phosphorylation of eIF2α. The ATF4 transcriptional program consists of a diverse set of genes with cytoprotective function, but chronic activation induces apoptosis indirectly through transcription of the mammalian ATF4 target CHOP. Yet, ATF4 activation has been detected in several human tumors, especially in hypoxic or nutrient-deprived regions, where ATF4 has been attributed with pro-survival and pro-proliferative effects. Interestingly, a recent study showed that melanoma cells respond to inhibition of their glycolytic metabolism by activating an ATF4 response, whose metabolic reconfiguration allows these cells to continue oncogenic growth, together arguing that ATF4 can provide cancer cells with a metabolic flexibility that allows them to tolerate hypoxic and nutritional stress or cancer therapy aimed at metabolism. Among the many conditions activating ATF4, recent work with cultured cells showed that inhibition of mitochondrial function is linked to ATF4 translation and activity. However, from these and other studies, both the mechanistic basis and the in vivo implications of this response remain to be elucidated (Sorge, 2020).

This study shows that in the fruit fly, Drosophila melanogaster, genetic perturbation of the electron transport chain (ETC), which induces a Warburg-like metabolism, activates a transcriptional stress response mediated through the eIF2α-kinase PERK and ATF4 in eye progenitor cells of Drosophila larvae. Importantly, this in vivo stress response is activated under ETC knockdown conditions, in the absence of obvious mitochondrial dysfunction. Interestingly, these results show that the ATF4 transcriptional response, which by itself causes reduced fitness of progenitor cells, is hijacked by growth-promoting pathways like Notch or Ras, leading to increased cellular fitness and enhanced proliferation. The data furthermore suggest that the pH changes associated with ETC impairment resulting in a switch of the metabolism to aerobic glycolysis play an important role in progenitor over-proliferation. In sum, this study shows that ATF4-mediated transcriptional adaptation provides a cell-autonomous response to ETC defects, altering cellular behavior through metabolic adaptation (Sorge, 2020).

Genetically induced disturbance of ETC complex assembly resultrd in a metabolic shift typical for mitochondrial impairment and activated an ATF4-dependent stress response. The in vivo transcriptional adaptation presented in this study confirmed the regulation of LDH and glycolytic enzymes, as shown in Drosophila cultured cells, and further includes several targets shown to be ATF4 target genes in mammalian models. The results showed that the eIF2α-kinase PERK, so far only described for its role in mediating one branch of the unfolded protein response of the endoplasmic reticulum (UPRER), is the upstream kinase phosphorylating eIF2α, thereby inducing ATF4 translation in response to mitochondrial ETC disturbance. Mitochondrial ETC disturbance specifically activated PERK, while other branches of the UPRER were non-responsive. PERK activation upon mitochondrial defects was recently observed in Drosophila models of Parkinson's disease and was explained by the authors by its preferential localization to mitochondria-associated ER membranes, which might make PERK more susceptible to a local stress signal. ROS (reactive oxygen species) released by mitochondria have been suggested to mediate mitochondrial retrograde signaling. While this study observed an attenuation of Delta overexpression (DlOE), COX7RNAi-induced over-proliferation upon overexpression of either cytoplasmic catalase or GPx (but not mitochondrial catalase), this study failed to detect increased ROS levels in the larval eye disc. A possible scenario to explain these observations is that ROS are generated locally in the cytoplasm or ER in response to ETC disturbance, thereby triggering PERK activation. Importantly, Drosophila PERK isoform B contains a potential mitochondrial signal peptide, which is not found in mammalian PERK isoforms. Although no evidence for this has been found, Drosophila PERK could reside in the mitochondrial membrane and sense the folding status of mitochondrial complexes. This hypothesis could explain the evolutionary difference between mitochondrial defects and ATF4 induction, as this appears to require GCN2 but not PERK in mammals or to be triggered independently of a single eIF2α-kinase. In addition to canonical ATF4 target genes, ATF4-dependent upregulation of mitochondrial chaperones, a response classically referred to as the mitochondrial UPR (UPRmt) was observed. In C. elegans, mitochondrial chaperone induction upon stress is mediated by ATF4-like transcription factor Atfs-1, while the mammalian UPRmt has been shown to be regulated by another evolutionary-related transcription factor, ATF5. The current data now showed that Drosophila ATF4 is required cell autonomously for the induction of mitochondrial chaperones upon ETC subunit knockdown, implying that Drosophila might represent the evolutionary ancestral ISR-UPRmt regulation through a single ATF4-like transcription factor (Sorge, 2020).

The cooperation between ATF4 target genes and the Notch or Ras pathways in Drosophila imaginal progenitors raised the intriguing possibility that these or other oncogenic pathways could benefit from ATF4 activity in human cancers. Over the last decades, it had been demonstrated that human cancer cells are exposed to several stresses, including hypoxia, ROS, or limitations in nutrient availability. In order to survive these conditions and maintain their growth capacity, tumor cells activate responses like the HIF1α transcription factor axis. Though less well studied, an involvement of ATF4 in cancer has been suggested mostly through work with cultured cells. This study analyzed gene expression in human cancer samples of The Cancer Genome Atlas (TCGA) datasets using Cancer-RNaseq-Nexus and the human protein pathology atlas and found that many of the well-characterized direct ATF4 targets are upregulated in a variety of cancer types. Most strikingly, transcriptomes of kidney renal clear cell carcinoma showed progressive induction of ATF4 and many of its direct targets (EIF4EBP1, ASNS, TRIB3, and VEGFA) on the transcriptional level, which strongly correlated with a poor prognosis in this type of cancer. These data suggest that the ATF4-mediated ISR is used by cancer cells to adapt their metabolic repertoire, thereby sustaining fast growth under increasingly unfavorable conditions (Sorge, 2020).

A novel finding presented in this study was the discovery that ATF4-mediated transcriptional adaptation due to ETC impairment allowed eye progenitors to increase their proliferation in response to signals from the Notch and Ras pathways. The primary questions arising from this genetic interaction is how these signaling pathways can overcome the apparent cellular stress and reduction in proliferation and induce the opposite effect, a massively increased rate of proliferation. Several lines of evidence suggest that over-proliferation in DlOE, COX7RNAi eye imaginal discs is controlled by pH changes induced by LDH that modify the activity of Notch downstream effectors. First, in COX7a-depleted cells, the metabolism is switched to aerobic glycolysis, leading to an increased production of lactate due to the activity of LDH. And, consistent with an accumulation of this metabolic acid, this study found the intracellular pH to be reduced in COX7RNAi cells. Second, DlOE, COX7RNAi-mediated over-proliferation was rescued by ATF4 knockdown and, to a lesser extent, pH buffering, showing that intracellular pH changes (downstream of ATF4 and LDH) play an important role in proliferation control. Third, LDH phenocopies COX7RNAi, indicating that most of the cooperative effects of Dl overexpression and COX7a knockdown are mediated by the ATF4 target LDH. In the same line, expression of LDH as one of the many ATF4 targets was sufficient to drive Dl-expressing cells into over-proliferation, strongly suggesting that the processes downstream of LDH-in particular, the changes in intracellular pH-lead to a modification of the Notch pathway. Finally, a cooperation of Notch and the COX7a nuclear effector ATF4 on the transcriptional level was not observed, showing that the Notch pathway is not hyper-activated, but arguing that over-proliferation is due to changes in the activity of Notch downstream effectors. The next obvious question is how changes in intracellular pH can modify the activity of signaling pathways. It is known that the intracellular pH can control the protonation of specific histidine residues in proteins acting as pH sensors, leading to changes in protein properties. Importantly, it has been shown recently in chicken embryos that intracellular pH changes induced by a Warburg-like metabolism control the acetylation of the Wnt effector &betsa;-catenin, thereby mediating Wnt signaling activation. Thus, this study envisions that pH changes induced by LDH expression lead to a (non-enzymatic) modification of Notch effectors, thereby increasing fitness and proliferation rates of eye progenitor cells (Sorge, 2020).

This is a very attractive model; however, one result was puzzling. Although LDH is sufficient to induce over-proliferation when combined with the Notch pathway, no rescue (but an increase in the severity) of the DlOE, COX7RNAi phenotype was observed when LDH was selectively depleted. This obvious discrepancy can be explained by different hypotheses. One of them is based on a recent study showing that LDHA inhibition in melanoma cell lines also failed to impact cell proliferation, survival, or tumor growth. In this context, LDHA inhibition engaged the GCN2-ATF4 signaling axis to initiate an expansive pro-survival response, including the upregulation of the glutamine transporter SLC1A5 and glutamine uptake, as well as mTORC1 activation. Another hypothesis is based on the finding that a major driver of over-proliferation is the intracellular pH. Since LDH catalyzes the conversion of pyruvate to lactate (and back), reducing LDH levels will affect the ratio of lactate to pyruvate, leading to an increase of the even stronger metabolic acid pyruvate. It has been shown that pyruvate as lactate induces a concentration-dependent intracellular acidification. Thus, it could be envisioned that an enhancement of proliferation rates beyond those observed in DlOE, COX7RNAi cells is a consequence of pyruvate accumulation in the absence of LDH, which enhances the decrease in the intracellular pH, resulting in the increase in proliferation rates (Sorge, 2020).

Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. This study shows that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. It is proposed that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes (Medina-Yanez, 2020).

Distant activation of Notch signaling induces stem cell niche assembly

This study shows that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, the formation was studied of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine (Yatsenko, 2021).

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. This study reports that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions (Sood, 2022).

This study reports that Notch signaling regulates quiescence, entry and exit in Drosophila central brain (CB) NBs. Increasing Notch pathway activity induces CB NBs to exit cell cycle via a Dap-dependent mechanism. Dap, a cyclin-dependent kinase inhibitor and CIP/KIP family member, is a known Notch target gene as is Cyclin E, String (Cdc25) and E2F. Whether Notch regulates other cell cycle genes required for CB NB exit remains unknown. Once CB NBs stop dividing, Notch pathway activity becomes attenuated. Low to no Notch activity is required for CB NBs to exit quiescence in response to dietary nutrient cues. Thus, levels of CB NB Notch activity regulate both the entry and exit from quiescence. High Notch is required for entry, whereas low Notch is required for exit. Intestinal stem cells (ISCs) also experience a period of low to no Notch activity during mid-pupal stages and, if Notch is ectopically induced at this time, ISCs terminally differentiate into secretory enteroendocrine cells. Whether any of the quiescent CB NBs with ectopic Notch terminally differentiate remains unknown (Sood, 2022).

Although Notch is required for quiescence, most CB NBs do stop dividing, albeit late. This suggests that other genes or signaling pathways are required or that residual Notch activity is sufficient to induce CB NB quiescence. Unfortunately, Notch null mutants are embryonic lethal. In addition, this study has shown that Notch is sufficient to induce quiescence. Although quiescence occurs prematurely, it is still restricted to late embryonic stages. Restriction of Notch function (cell cycle exit) is likely due to CB NB intrinsic temporal programs. Temporal programs that likely vary across CB NB lineages, as is the case in the ventral nerve cord. Whether temporal programs regulate levels of Notch pathway activity or provide additional factors needed for CB NB cell cycle exit remains unanswered (Sood, 2022).

In mammals, Notch signaling is more complicated because of gene duplication. Yet, recently, Notch signaling has been shown to regulate neural stem cell quiescence. In Notch2 conditional knockout mice, more neural stem cells are actively dividing in the hippocampus and subventricular zone brain regions in adult animals compared with controls. This results in premature depletion of the neural stem pool and reduced neurogenesis in older mice. This is similar to what is reported in this study and, in the future, it will be interesting to determine which Notch ligands are required and whether neural stem cells in mammals use their newborn daughters for pathway activation (Sood, 2022).

Genetic interactions regulate hypoxia tolerance conferred by activating Notch in excitatory amino acid transporter 1-positive glial cells in Drosophila melanogaster

Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. This study delineates the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. The role of a group of conserved genes was determined that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). A novel dual-UAS/Gal4 system was developed that allows activation of Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, it was discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Because these genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases (Zhou, 2021)

Membrane architecture and adherens junctions contribute to strong Notch pathway activation

The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing the investigation of the importance of membrane architecture and specific junctional domains for signaling. By measuring Notch dependent transcription in live embryos it was established that Notch initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junction likely play an important role in modulating Notch activity (Falo-Sanjuan, 2021).

Geometry of gene regulatory dynamics

Embryonic development leads to the reproducible and ordered appearance of complexity from egg to adult. The successive differentiation of different cell types that elaborate this complexity results from the activity of gene networks and was likened by Waddington to a flow through a landscape in which valleys represent alternative fates. Geometric methods allow the formal representation of such landscapes and codify the types of behaviors that result from systems of differential equations. Results from Smale and coworkers imply that systems encompassing gene network models can be represented as potential gradients with a Riemann metric, justifying the Waddington metaphor. This study extends the representation to include parameter dependence and enumerate all three-way cellular decisions realizable by tuning at most two parameters, which can be generalized to include spatial coordinates in a tissue. All diagrams of cell states vs. model parameters are thereby enumerated. The study unifies a number of standard models for spatial pattern formation by expressing them in potential form (i.e., as topographic elevation). Turing systems appear nonpotential, yet in suitable variables the dynamics are low dimensional and potential. A time-independent embedding recovers the original variables. Lateral inhibition is described by a saddle point with many unstable directions. A model for the patterning of the Drosophila eye appears as relaxation in a bistable potential. Geometric reasoning provides intuitive dynamic models for development that are well adapted to fit time-lapse data (Rand, 2021).

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). This study tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. These findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation (Kuang, 2021).

Genetic and Molecular Interactions between H(ΔCT), a Novel Allele of the Notch Antagonist Hairless, and the Histone Chaperone Asf1 in Drosophila melanogaster

Cellular differentiation relies on the highly conserved Notch signaling pathway. Notch activity induces gene expression changes that are highly sensitive to chromatin landscape. This study addressed Notch gene regulation using Drosophila as a model, focusing on the genetic and molecular interactions between the Notch antagonist Hairless and the histone chaperone Asf1. Earlier work implied that Asf1 promotes the silencing of Notch target genes via Hairless (H). A novel ΔCT) allele was generated by genome engineering. Phenotypically, ΔCT behaves as a Hairless gain of function allele in several developmental contexts, indicating that the conserved CT domain of H has an attenuator role under native biological contexts. Using several independent methods to assay protein-protein interactions, this study defined the sequences of the CT domain that are involved in Hairless-Asf1 binding. Based on previous models, where Asf1 promotes Notch repression via Hairless, a loss of Asf1 binding should reduce Hairless repressive activity. However, tissue-specific Asf1 overexpression phenotypes are increased, not rescued, in the HΔCT background. Counterintuitively, Hairless protein binding mitigates the repressive activity of Asf1 in the context of eye development. These findings highlight the complex connections of Notch repressors and chromatin modulators during Notch target-gene regulation and open the avenue for further investigations (Maier, 2023).

Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells

In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. this study reports the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. This study proposes that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling (Houssin, 2021).

Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila

The adult Drosophila intestinal epithelium must be tightly regulated to maintain regeneration and homeostasis. The dysregulation of the regenerative capacity is frequently associated with intestinal diseases such as inflammation and tumorigenesis. This study shows that the G protein-coupled receptor Anchor maintains Drosophila adult midgut homeostasis by restricting Jun-N-terminal kinase (JNK) and Notch pathway activity. anchor inactivation resulted in aberrant JNK pathway activation, which led to excessive enteroblast (EB) production and premature enterocyte (EC) differentiation. In addition, increased Notch levels promoted premature EC differentiation following the loss of anchor. This defect induced by the loss of anchor ultimately caused sensitivity to stress or environmental challenge in adult flies. Taken together, these results demonstrate that the activity of anchor is essential to coordinate stem cell differentiation and proliferation to maintain intestinal homeostasis (Wang, 2021).

The expression of Catsup in escort cells affects Drosophila ovarian stem cell niche establishment and germline stem cells self-renewal via Notch signaling

Stem cell niche provides extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Drosophila ovarian stem cell niche is composed of somatic terminal filament cells, cap cells and escort cells. However, the underlying mechanism for the development of stem cell niche remains largely unclear. This study found that the expression of a zinc transporter Catsup is essential for ovary morphogenesis. Catsup knockdown in escort cells results in defects of niche establishment and germline stem cells self-renewal. These defects could be modified by altered expression of genes involved in zinc metabolism or intervention of dietary zinc levels. Further studies indicated that Catsup RNAi affected adult ovary morphogenesis by suppressing Notch signaling. Lastly, this study demonstrated that the defects of Catsup RNAi could be restored by overexpression of heat shock cognate protein 70 (Hsc70). These findings expand understanding of the mechanisms controlling adult oogenesis and niche establishment in Drosophila (Gao, 2023).

Identification of a new allele of O-fucosyltransferase 1 involved in Drosophila intestinal stem cell regulation

Adult stem cells are critical for the maintenance of tissue homeostasis. However, how the proliferation and differentiation of intestinal stem cells (ISCs) are regulated remains not fully understood. This study found a mutant, stum 9-3, affecting the proliferation and differentiation of Drosophila adult ISCs in a forward genetic screen for factors regulating the proliferation and differentiation ISCs. stum 9-3 acts through the conserved Notch signaling pathway, upstream of the S2 cleavage of the Notch receptor. Interestingly, the phenotype of stum 9-3 mutant is not caused by disruption of stumble (stum), where the p-element is inserted. Detailed mapping, rescue experiments and mutant characterization show that stum 9-3 is a new allele of O-fucosyltransferase 1 (O-fut1). These results indicate that unexpected mutants with interesting phenotype could be recovered in forward genetic screens using known p-element insertion stocks (Shi, 2021).

Repression of differentiation genes by Hes transcription factors fuels neural tumour growth in Drosophila

Neural stem cells (NSC) divide asymmetrically to generate a cell that retains stem cell identity and another that is routed to differentiation. Prolonged mitotic activity of the NSCs gives rise to the plethora of neurons and glial cells that wire the brain and nerve cord. Genetic insults, such as excess of Notch signaling, perturb the normal NSC proliferation programs and trigger the formation of NSC hyperplasias, that can later progress to malignancies. Hes proteins are crucial mediators of Notch signaling and in the NSC context they act by repressing a cohort of early pro-differentiation transcription factors. Downregulation of these pro-differentiation factors makes NSC progeny cells susceptible to adopting an aberrant stem cell program. It has been recently shown that Hes overexpression in Drosophila leads to NSC hyperplasias that progress to malignant tumours after allografting to adult hosts. This study combined genetic analysis, tissue allografting and transcriptomic approaches to address the role of Hes genes in NSC malignant transformation. The E(spl) genes are important mediators in the progression of Notch hyperplasias to malignancy, since allografts lacking the E(spl) genes grow much slower. RNA profiling is presented of Hes-induced tumours at two different stages after allografting. The same cohort of differentiation-promoting transcription factors that are repressed in the primary hyperplasias continue to be downregulated after transplantation. This is accompanied by an upregulation of stress-response genes and metabolic reprogramming. It is concluded that the combination of dedifferentiation and cell physiology changes most likely drive tumour growth (Voutyraki, 2021).

The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand-receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. EP555 was identified as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which are accordingly renamed Membrane-bound Notch regulator (Mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand-receptor interaction. Evidence is provided that Mnr plays a role in &gamms;-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. It is concluded that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, a membrane-bound factor has been identified that directly augments Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity (Nagel, 2021).

Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors

CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. This study evaluated the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. Conditional tissue-specific CRISPR knockouts were generated using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, regional differences were observed in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. These findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo (Bahuguna, 2021).

Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway

Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. This study analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. A analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. Two functions were identifiee for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. These findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms (Schnute, 2022).

Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs), are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in twelve hours during midoogenesis. Previously, it was reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. This study reports that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for the SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study single-cell RNA sequencing data was used to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened (Jia, 2022).

A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects. PLoS Genet 18(8): e1010335

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. This study found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation (Gagliani, 2022).

ASC proneural factors are necessary for chromatin remodeling during neuroectodermal to neuroblast fate transition to ensure the timely initiation of the neural stem cell program

In both Drosophila and mammals, the achaete-scute (ASC/ASCL) proneural bHLH transcription factors are expressed in the developing central and peripheral nervous systems, where they function during specification and maintenance of the neural stem cells in opposition to Notch signaling. However, the impact of ASC on chromatin dynamics during neural stem cell generation remains elusive. This study investigated the chromatin changes accompanying neural commitment using an integrative genetics and genomics methodology. ASC factors were found to bind equally strongly to two distinct classes of cis-regulatory elements: open regions remodeled earlier during maternal to zygotic transition by Zelda and less accessible, Zelda-independent regions. Both classes of cis-elements exhibit enhanced chromatin accessibility during neural specification and correlate with transcriptional regulation of genes involved in a variety of biological processes necessary for neuroblast function/homeostasis. This study identified an ASC-Notch regulated TF network that includes likely prime regulators of neuroblast function. Using a cohort of ASC target genes, it is reported that ASC null neuroblasts are defectively specified, remaining initially stalled, unable to divide, and lacking expression of many proneural targets. When mutant neuroblasts eventually start proliferating, they produce compromised progeny. Reporter lines driven by proneural-bound enhancers display ASC dependency, suggesting that the partial neuroblast identity seen in the absence of ASC genes is likely driven by other, proneural-independent, cis-elements. Neuroblast impairment and the late differentiation defects of ASC mutants are corrected by ectodermal induction of individual ASC genes but not by individual members of the TF network downstream of ASC. However, in wild-type embryos, the induction of individual members of this network induces CNS hyperplasia, suggesting that they synergize with the activating function of ASC to consolidate the chromatin dynamics that promote neural specification. This study has demonstrate that ASC proneural transcription factors are indispensable for the timely initiation of the neural stem cell program at the chromatin level by regulating a large number of enhancers in the vicinity of neural genes. This early chromatin remodeling is crucial for both neuroblast homeostasis as well as future progeny fidelity (Theodorou, 2022).

Protein kinase CK2 phosphorylates a conserved motif in the Notch effector E(spl)-Mgamma

Across metazoan animals, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although these repressors are generally conserved, their sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the Orange (O) domain from the penultimate WRPW tetrapeptide motif that binds the obligate co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, bioinformatics, evolutionary, and biochemical analyses were conducted. These studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-β subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50 × 10(6) years of Drosophila evolution. Thus, a preponderance of E(spl) homologues (four out of seven total) in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins (Jozwick, 2022).

MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine

Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood. This study reports that commitment to the terminally differentiated enterocyte (EC) cell fate is under microRNA control. An intestinally enriched microRNA, miR-956, fine-tunes Notch signaling activity specifically in intermediate, enteroblast (EB) progenitor cells to control EC differentiation. This study further identified insensitive mRNA as a target of miR-956 that regulates EB/EC ratios by repressing Notch activity in EBs. In summary, this study highlights a post-transcriptional gene-regulatory mechanism for controlling differentiation in an adult intestinal stem cell lineage (Mukherjee, 2022).

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. In the absence of ligand, a negative regulatory region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site; ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction. In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a "pulling force" on Notch that is essential to expose the cleavage site in the NRR. This study shows that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila, evidence is presented that (1) the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a "leucine plug" that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs. These results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant (Langridge, 2022).

The mitochondrial ribosomal protein mRpL4 regulates Notch signaling

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. This study reports a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. mRpL4 physically and genetically interacts with the WD40 repeat protein Wings apart (Wap) and activates the transcription of Notch signaling targets. This study shows that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, this study has discovered a previously unknown function of mRpL4 during animal development (Mo, 2023).

A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla

Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts are temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with the generation of the appropriate number of neurons at each stage. This study used neuroblasts of the Drosophila optic lobe medulla to address these questions and show that the expression of TTFs Sloppy-paired 1/2 (Slp1/2) is directly regulated at the transcriptional level by two other TTFs and the cell-cycle dependent Notch signaling through two cis-regulatory elements. It was also shown that supplying constitutively active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. These findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade (Ray, 2022).

Drosophila neuroblasts are temporally patterned by sequentially expressed TTFs. Although the expression pattern and mutant phenotypes suggest that TTFs form a transcriptional cascade, direct transcriptional regulation between TTFs has not been demonstrated in most cases. This work has characterized two enhancers of the slp genes that enable the expression of Slp1 and Slp2 in medulla neuroblasts. The u8772 220 bp enhancer is activated at an earlier stage relative to the d5778 850 bp enhancer. In these two enhancers, sites were identified for the previous TTF -Ey and Scro-a TTF expressed at around the same time as Slp1. Deleting either enhancer alone did not eliminate the expression of endogenous Slp1 and Slp2, suggesting that they act partially redundantly with one another. Deletion of both enhancers completely eliminates Slp1 and Slp2 expression in medulla neuroblasts but does not affect their expression in lamina neurons or glia, confirming the specificity and necessity of these two enhancers. Using GFP reporter assays, this study has shown that mutation of Ey binding sites in these enhancers abolishes reporter expression similar to genetic experiments where a loss of GFP reporter was reported within ey RNAi clones. The results are also consistent with previous studies that showed a complete loss of endogenous Slp1/2 expression in UAS-ey-RNAi expressing neuroblasts. This study also confirmed the in vivo binding of Ey to the identified enhancers of Slp by Dam-ID sequencing. The expression of the TTF Scro is initiated simultaneously as Slp1/2, and it has been shown that loss of Scro significantly reduces Slp expression level. Mutation of most probable Scro binding sites on the u8772 220 bp or d5778 850 bp enhancers led to a dramatic reduction of GFP reporter expression. Thus, the combined effect of mutating Scro binding sites on both enhancers recapitulates the observed impact of Scro knock-down on endogenous Slp1/2 expression, which is reduced expression of Slp1/2 in neuroblasts expressing UAS scro-RNAi and a consequent loss of neural fates specified by Slp1/2 in their progeny. It is interesting to note that the observation of multiple enhancers regulating Slp1/2 expression is consistent with regulation of Slp1/2 in other developmental contexts. Previous studies have noted the presence of multiple enhancers of Slp1/2 expression in the vicinity of the slp1 and slp2 coding loci. Many of these regulatory DNA segments function as stripe enhancers enabling Slp1/2 to function as pair-rule genes during embryonic segmentation. Although these enhancers share some overlapping functions and domains of activation, a full complement of stripe enhancers is required for maintaining parasegment boundaries and wingless expression (Ray, 2022).

It was previously demonstrated that although Ey is necessary for activating Slp1/2 expression it is not sufficient. There is always a time delay after the start of Ey expression to the start of Slp expression to ensure the sufficient duration of the Ey window. How is the timing controlled? From analyses of the slp1/2 enhancer sequences, several binding sites were found for the CSL transcription factor Su(H), most prominently known as a central component of the ternary Notch transcription complex and the primary DNA binding component. To confirm the involvement of the Notch pathway in regulating Slp1/2, we observed the effects of knocking down key Notch pathway components on endogenous Slp1/2 expression. In all cases a delay was observed in the expression of Ey and a further delay in the transition to the Slp1/2 stage in neuroblasts expressing the RNAi knockdowns. Mutating Su(H) binding sites in the u8772 220bp enhancer led to a loss or reduction of GFP reporter expression in neuroblasts. However, mutating Su(H) binding sites in the d5778 850 bp enhancer did not decrease the reporter expression. These results suggest that Notch signaling directly regulates Slp expression through the u8772 220 bp enhancer, but not the d5778 850 bp enhancer, and this is consistent with the delayed expression driven by the d5778 850 bp enhancer. However, Ey still plays a more critical role in activating Slp1/2 expression than the Notch pathway, since Slp1/2 are still expressed albeit later in the absence of Su(H) and other Notch components, and Notch signaling requires Ey to speed up the Ey to Slp transition. As with Ey, this study confirmed Su(H) binding to the u872 220 bp enhancer using DamID-seq. Thus, this work provided strong evidence that N signaling, a general signaling pathway involved in neuroblast development, regulates the timing of activation of a TTF gene directly. In addition, the results also raised the interesting hypothesis that Notch signaling might be involved in facilitating all temporal transitions, because the turning on of Opa and Ey is also delayed, and a further and further delay was observed in turning on of later TTFs. Whether Notch signaling regulates other TTF expression directly or indirectly still awaits further investigation. (Ray, 2022).

What might explain the delay in Slp1/2 expression in the absence of Notch signaling? Recent developments in single-molecule Fluorescence In Situ Hybridization (smFISH) technology and live imaging techniques using the MS2-MCP system have enabled studying the transcription process in molecular detail. Imaging transcription driven by Notch responsive enhancers in native contexts has shown this process to be inherently 'bursty', i.e., episodes of transcription (enhancer 'On' state) are punctuated with gaps in activity (enhancer in 'Off' state) (Falo-Sanjuan et al., 2019; Lee et al., 2019). The dosage of NICD modulates the duration of the 'On' phase in one context studied by live imaging (Falo-Sanjuan et al., 2019; Lee et al., 2019). Additionally, binding of tissue-specific regional factors to these Notch responsive enhancers may prime these enhancers and help synchronize transcription and sustain a steady transcriptional output upon Notch binding to enhancers; this helps integrate important positional cues and the perception of context (Falo-Sanjuan et al., 2019). Applying these insights to our system, we suggest that Ey may act by priming the Notch-responsive enhancer of slp providing crucial contextual information, and this is required for Notch to further activate Slp1/2 transcription, and speed up the transition. (Ray, 2022).

Notch target genes and Dpn are transcriptional repressors that act partially redundantly to maintain neuroblast identity. In type II NBs, Dpn depends on Notch signaling, and loss of Dpn causes premature differentiation. However, in type I NBs, Dpn is not lost when Notch signaling is lost, and Notch signaling seems dispensable for the self-renewing abilities of NBs. In the medulla neuroblasts, this study also observed that in Su(H) mutant clones, the clone size and neuroblast proliferation are not significantly affected. On the other hand, N signaling was observed to be dependent on cell-cycle progression, and the Notch target gene is lost when cell cycle progression is blocked. (Ray, 2022).

In the medulla, blocking cell cycle progression in neuroepithelial cells prematurely transforms them into neuroblasts, and these neuroblasts seem to be arrested or severely delayed in the TTF cascade. When the cell cycle was arrested or slowed down later in neuroblasts to preserve Ey expression, Slp expression was is still delayed. Therefore, cell cycle progression also has a role in the Ey to Slp transition. Further, this study showed that supplying Notch signaling is sufficient to rescue the delay in the Ey to Slp transition caused by cell cycle defect. Thus at the Ey to Slp transition, the cell cycle effect is mediated through the direct regulation of Slp transcription by Notch signaling. Taken together, these results suggest that in Ey stage neuroblasts, Ey is required to initiate Slp expression but not sufficient to activate it to a strong level right away, and after each asymmetric division, activation of Notch signaling in the neuroblast enhances Slp expression, until Slp expression reaches a certain level to repress Ey expression and make the transition. This can be part of a mechanism to coordinate the TTF temporal transition with the cell cycle progression to generate the appropriate number of neural progenies at a given temporal stage (Ray, 2022).

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).

This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).

The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).

Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).

Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).

Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).

dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).

As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).

In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).

The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation, with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context. The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).

Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).

N-cad mutant ommatidia appear to over-rotate unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).

The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).

The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).

The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).

There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).

Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells. It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).

Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argosrlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).

Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).

Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos, with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR. This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).

Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).

This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).

The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).

list of proneural and neurogenic genes


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Schnute, B., Shimizu, H., Lyga, M., Baron, M. and Klein, T. (2022). Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway. BMC Biol 20(1): 65. PubMed ID: 35264151

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Zygotically transcribed genes

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