The Interactive Fly

Zygotically transcribed genes

Proneural and Neurogenic Genes

What is the Notch pathway?

Developmental biology of the Notch pathway

The achaete-scute complex

Embryonic central nervous system lineages

Aging neural progenitors lose competence to respond to mitogenic Notch signaling

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

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

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

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

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

Neurogenic genes functioning in the Notch pathway

Proneural genes of the achaete-scute complex

Additional proneural genes

What is the Notch pathway?

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

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

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

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

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

Developmental biology of the Notch pathway

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

The achaete-scute complex

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

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

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

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

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

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

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

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

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

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

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

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

Embryonic central nervous system lineages

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

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

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

Aging neural progenitors lose competence to respond to mitogenic Notch signaling

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

list of proneural and neurogenic genes


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Zygotically transcribed genes

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