Proneural Clusters Research Articles

Genetic Programs Activated by Proneural Proteins in the Developing Drosophila PNS

Neurogenesis depends on a family of proneural transcriptional activator proteins, but the "proneural" function of these factors is poorly understood, in part because the ensemble of genes they activate, directly or indirectly, has not been identified systematically. We have taken a direct approach to this problem in Drosophila. Fluorescence-activated cell sorting was used to recover a purified population of the cells that comprise the "proneural clusters" from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Bioinformatic and reporter gene studies further illuminate the cis-regulatory codes that direct expression in proneural clusters.

Charlatan, a Zn-finger transcription factor, establishes a novel level of regulation of the proneuralachaete/scutegenes ofDrosophila

The proneural genes achaete (ac) and scute (sc) are necessary for the formation of the external sensory organs (SOs) of Drosophila. ac and sc are expressed in proneural clusters and impart their cells with neural potential. For this potential to be realized, and the SO precursor cell (SOP) to arise within a cluster, sufficient proneural protein must accumulate in the cluster. Here we describe a novel gene, charlatan (chn), which encodes a zinc finger transcription factor that facilitates this accumulation by forming a stimulatory loop with ac/sc. We find that loss of function of chn decreases the accumulation of Sc in proneural clusters and partially removes notum macrochaetae, while overexpression of chn enhances ac/sc expression and the formation of extra SOs. Moreover, chn is activated by ac/sc in proneural clusters. Chn apparently stimulates ac/sc by physically interacting with the proneural cluster-specific enhancers and increasing enhancer efficiency, thus acting as a stimulator of ac/sc expression in proneural clusters. chn is also required for the proper development of the embryonic peripheral nervous system, as its absence leads to loss of neurons and causes aberrant development of chordotonal organs.

A DNA Transcription Code for Cell-Specific Gene Activation by Notch Signaling

Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or "architecture" within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to "S" DNA sites and proneural bHLH activator proteins bound to nearby "A" DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an "S+A" transcription code) can mediate the Notch-proneural transcriptional synergy. By contrast, we show that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ("SPS"), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, "SPS+A", that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. We also present evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. The SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression.

Drosophila CK2 regulates eye morphogenesis via phosphorylation of E(spl)M8

The Notch effector E(spl)M8 is phosphorylated at Ser 159 by CK2, a highly conserved Ser/Thr protein kinase. We have used the Gal4-UAS system to assess the role of M8 phosphorylation during bristle and eye morphogenesis by employing a non-phosphorylatable variant (M8SA) or one predicted to mimic the ‘constitutively’ phosphorylated protein (M8SD). We find that phosphorylation of M8 does not appear to be critical during bristle morphogenesis. In contrast, only M8SD elicits a severe ‘reduced eye’ phenotype when it is expressed in the morphogenetic furrow of the eye disc. M8SD elicits neural hypoplasia in eye discs, elicits loss of phase-shifted Atonal-positive cells, i.e. the ‘founding’ R8 photoreceptors, and consequently leads to apoptosis. The ommatidial phenotype of M8SD is similar to that in N spl/ Y; E( spl) D/+ flies. E( spl) D , an allele of m 8, encodes a truncated protein known as M8*, which, unlike wild type M8, displays exacerbated antagonism of Atonal via direct protein–protein interactions. In line with this, we find that the M8SD–Atonal interaction appears indistinguishable from that of M8*-Atonal, whereas interaction of M8 or M8SA appears marginal, at best. These results raise the possibility that phosphorylation of M8 (at Ser 159) might be required for its ability to mediate ‘lateral inhibition’ within proneural clusters in the developing retina. This is the first identification of a dominant allele encoding a phosphorylation-site variant of an E(spl) protein. Our studies uncover a novel functional domain that is conserved amongst a subset of E(spl)/Hes repressors in Drosophila and mammals, and suggests a potential role for CK2 during retinal patterning.

Echinoid synergizes with the Notch signaling pathway inDrosophilamesothorax bristle patterning

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. We show a novel function of Ed, i.e. the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. We conclude that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, we find that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway.

Delta-promoted filopodia mediate long-range lateral inhibition in Drosophila.

Drosophila thoracic mechanosensory bristles originate from cells that are singled out from 'proneural' groups of competent epithelial cells. Neural competence is restricted to individual sensory organ precursors (SOPs) by Delta/Notch-mediated 'lateral inhibition', whereas other cells in the proneural field adopt an epidermal fate. The precursors of the large macrochaetes differentiate separately from individual proneural clusters that comprise about 20-30 cells or as heterochronic pairs from groups of more than 100 cells, whereas the precursors of the small regularly spaced microchaetes emerge from even larger proneural fields. This indicates that lateral inhibition might act over several cell diameters; it was difficult to reconcile with the fact that the inhibitory ligand Delta is membrane-bound until the observation that SOPs frequently extend thin processes offered an attractive hypothesis. Here we show that the extension of these planar filopodia--a common attribute of wing imaginal disc cells--is promoted by Delta and that their experimental suppression reduces Notch signalling in distant cells and increases bristle density in large proneural groups, showing that these membrane specializations mediate long-range lateral inhibition.

Senseless acts as a binary switch during sensory organ precursor selection.

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Here, we present evidence that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. We show that Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express low levels of Sens, it acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express high levels of Sens, it acts as a transcriptional activator and synergizes with proneural proteins. We therefore propose that Sens acts as a binary switch that is fundamental to SOP selection.

The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification.

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.

The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila.

In the Drosophila embryo, studies on CNS development have so far mainly focused on the relatively simply structured ventral nerve cord. In the trunk, proneural genes become expressed in small cell clusters at specific positions of the ventral neuroectoderm. A lateral inhibition process mediated by the neurogenic genes ensures that only one cell within each proneural cluster delaminates as a neural stem cell (neuroblast). Thus, a fixed number of neuroblasts is formed, according to a stereotypical spatiotemporal and segmentally repeated pattern, each subsequently generating a specific cell lineage. Owing to higher complexity and hidden segmental organisation, the mechanisms underlying the development of the brain are much less understood. In order to pave the way towards gaining deeper insight into these mechanisms, we have undertaken a comprehensive survey of early brain development until embryonic stage 11, when all brain neuroblasts have formed. We describe the complete spatiotemporal pattern of formation of about 100 brain neuroblasts on either side building the trito-, deuto- and protocerebrum. Using 4D-microscopy, we have uncovered various modes of neuroblast formation that are related to specific mitotic domains of the procephalic neuroectoderm. Furthermore, a detailed description is provided of the dynamic expression patterns of proneural genes (achaete, scute, lethal of scute, atonal) in the procephalic neuroectoderm and the individual neuroblasts. Finally, we present direct evidence that, in contrast to the trunk, adjacent cells within specific domains of the procephalic neuroectoderm develop as neuroblasts, indicating that mechanisms controlling neuroblast formation differ between head and trunk.

A Notch-independent function of Suppressor of Hairless during the development of the bristle sensory organ precursor cell of Drosophila.

Su(H)/CBF1 is a key component of the evolutionary conserved Notch signalling pathway. It is a transcription factor that acts as a repressor in the absence of the Notch signal. If Notch signalling is activated, it associates with the released intracellular domain of the Notch receptor and acts as an activator of transcription. During the development of the mechanosensory bristles of Drosophila, a selection process called lateral inhibition assures that only a few cells are selected out of a group to become sensory organ precursors (SOP). During this process, the SOP cell is thought to suppress the same fate in its surrounding neighbours via the activation of the Notch/Su(H) pathway in these cells. We show that, although Su(H) is required to prevent the SOP fate during lateral inhibition, it is also required to promote the further development of the SOP once it is selected. Importantly, in this situation Su(H) appears to act independently of the Notch signalling pathway. We find that loss of Su(H) function leads to an arrest of SOP development because of the loss of sens expression in the SOP. Our results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. We show that this repressor activity is encoded by one or several genes of the E(spl)-complex. Our results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl.

The Drosophila IgC2 domain protein friend-of-echinoid, a paralogue of echinoid, limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway

The Notch signaling pathway is critical in cell fate specification throughout development. In the developing wing disc, single sensory organ precursors (SOPs) are selected from proneural clusters via a process of lateral inhibition mediated by the Notch signaling pathway. The epidermal growth factor receptor (EGFR) pathway has also been implicated in SOP formation. Here, we describe the Drosophila melanogaster gene friend of echinoid (fred), a paralogue of echinoid (ed), a gene recently identified as a negative regulator of the EGFR pathway. fred function was examined in transgenic flies by using inducible RNA interference (RNAi). Suppression of fred in developing wing discs results in specification of ectopic SOPs, additional microchaeta, and cell death. In eye-antennal discs, fred suppression causes a rough eye phenotype. These phenotypes are suppressed by overexpression of Notch, Suppressor of Hairless [Su(H)], and Enhancer of split m7. In contrast, overexpression of Hairless, a negative regulator of the Notch pathway, and decreased Su(H) activity enhance these phenotypes. Thus, fred acts in close concert with the Notch signaling pathway. Dosage-sensitive genetic interaction also suggests a close relationship between fred and ed.

Interkinetic Nuclear Movement May Provide Spatial Clues to the Regulation of Neurogenesis

During the transition from S phase to mitosis, vertebrate neuroepithelial cells displace their nuclei and subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear movement (INM). Here we provide evidence that cycling neuroepithelial cells pass through a neurogenic state in which they are situated apically, as defined by the capacity to express Notch1, Delta1, and Neurogenin2 ( Ngn2). Based on this scenario, we have developed a mathematical model to analyze the influence of INM on neurogenesis. In the absence of INM, the model predicted an increase in the rate of neurogenesis due to the reduction in the influence of inhibitory signals on cells in the neurogenic state. This exacerbation in neurogenesis led to the diminished growth of the neuroepithelium and a reduction in the later production of neurons. Pharmacological perturbation of the stereotypical distribution of precursors along the orthogonal axis of the neuroepithelium resulted in an excess of neurogenesis, as seen by the expression of Ngn2, and of the neuronal marker RA4 in the retina. These findings suggest that INM might be important for the efficient and continued production of neurons in G0, since it is involved in defining a proneural cluster in the ventricular part of the neuroepithelium that contains precursors at stages of the mitotic cycle compatible with neuronal differentiation.

Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless.

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.

Tramtrack co-operates to prevent inappropriate neural development in Drosophila

Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete–Scute complex (AS–C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. We demonstrate that the BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, we dissected functional domains of Ttk, confirming the importance of the bric-à-brac–tramtrack–broad complex (BTB) motif. We show that the Ttk BTB domain is a protein–protein interaction motif mediating tetramer formation.

Hox gene control of segment-specific bristle patterns in Drosophila.

Hox genes specify the different morphologies of segments along the anteroposterior axis of animals. How they control complex segment morphologies is not well understood. We have studied how the Hox gene Ultrabithorax (Ubx) controls specific differences between the bristle patterns of the second and third thoracic segments (T2 and T3) of Drosophila melanogaster. We find that Ubx blocks the development of two particular bristles on T3 at different points in sensory organ development. For the apical bristle, a precursor is singled out and undergoes a first division in both the second and third legs, but in the third leg further differentiation of the second-order precursors is blocked. For the posterior sternopleural bristle, development on T3 ceases after proneural cluster initiation. Analysis of the temporal requirement for Ubx shows that in both cases Ubx function is required shortly before bristle development is blocked. We suggest that interactions between Ubx and the bristle patterning hierarchy have evolved independently on many occasions, affecting different molecular steps. The effects of Ubx on bristle development are highly dependent on the context of other patterning information. Suppression of bristle development or changes in bristle morphology in response to endogenous and ectopic Ubx expression are limited to bristles at specific locations.

A genetic hierarchy establishes mitogenic signalling and mitotic competence in the renal tubules of Drosophila.

Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signalling from a specialised subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to secrete the EGF ligand, Spitz. We show that the response to EGF signalling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signalling by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of pointedP2 is induced by Wingless signalling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. We demonstrate that both signalling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a two-step process. First, a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression of seven up. Second, lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete Spitz and become signalling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable response to mitogenic signalling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia.

Scute expression in Calliphora vicina reveals an ancestral pattern of longitudinal stripes on the thorax of higher Diptera.

In Drosophila the stereotyped arrangement of sensory bristles on the notum is determined by the tightly regulated control of transcription of the achaete-scute (ac-sc) genes which are expressed in small proneural clusters of cells at the sites of each future bristle. Expression relies on a series of discrete cis-regulatory elements present in the ac-sc gene complex that are the target of the transcriptional activators pannier (pnr) and the genes of the iroquois complex. Stereotyped bristle patterns are common among species of acalyptrate Schizophora such as Drosophila, and are thought to have derived from an ancestral pattern of four longitudinal rows extending the length of the scutum, through secondary loss of bristles. To investigate evolutionary changes in bristle patterns and ac-sc regulation by pnr, we have isolated homologues of these genes from Calliphora vicina, a species of calyptrate Schizophora separated from Drosophila by at least 100 million years. Calliphora vicina displays a pattern of four rows of bristles on the scutum resembling the postulated ancestral one. We find that sc in Calliphora is expressed in two longitudinal stripes on the medial scutum that prefigure the development of the rows of acrostichal and dorsocentral bristles. This result suggests that a stripe-like expression pattern of sc may be an ancestral feature and may have preceded the evolution of proneural clusters. The implications for the evolution of the cis-regulatory elements responsible for sc expression in the proneural clusters of Drosophila, and function of Pnr are discussed.

A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny.

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from a proneural cluster, and undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. We show that phyllopod (phyl), previously identified to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters, leading to the fate transformation of neurons and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. We further show that seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. It has been shown previously that Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. We further demonstrate that phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development.

Regulation of dally, an Integral Membrane Proteoglycan, and Its Function during Adult Sensory Organ Formation of Drosophila

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. Here, we show that dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. We have also determined that, for pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters.

Scabrous complexes with Notch to mediate boundary formation.

The mechanisms that establish and sharpen pattern across epithelia are poorly understood. In the developing nervous system, the first pattern elements appear as 'proneural clusters' In the morphogenetic furrow of the immature Drosophila retina proneural clusters emerge in a wave as a patterned array of 6-10-cell groups, which are recognizable by expression of Atonal, a basic helix-loop-helix transcription factor that is required to establish and pattern the first cell fate. The establishment and subsequent patterning of Atonal expression requires activity of the signalling transmembrane receptor Notch. Here we present in vivo and biochemical evidence that the secreted protein Scabrous associates with Notch, and can stabilize Notch protein at the surface. The result is a regulation of Notch activity that sharpens proneural cluster boundaries and ensures establishment of single pioneer neurons.