scabrous modifies epithelial cell adhesion and extends the range of lateral signalling during development of the spaced bristle pattern in Drosophila.

The role of scabrous (sca) in the evenly spaced bristle pattern of Drosophila is explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighbouring cells, causing them to adopt the epidermal fate. We show that Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signalling has been increased. scabrous acts nonautonomously, and we present evidence that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signalling are discussed. We also show that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and speculate on a possible role for these structures during signalling.     

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate.

BACKGROUND: The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS: Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS: Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.     

Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila

Cells in the neurectoderm of Drosophila face a choice between neural and epidermal fates. On the notum of the adult fly, neural cells differentiate sensory bristles in a precise pattern. Evidence has accumulated that the bristle pattern arises from the spatial distribution of small groups of cells, proneural clusters, from each of which a single bristle will result. One class of genes, which includes the genes of the achaete-scute complex, is responsible for the correct positioning of the proneural clusters. The cells of a proneural cluster constitute an equivalence group, each of them having the potential to become a neural cell. Only one cell, however, will adopt the primary, dominant, neural fate. This cell is selected by means of cellular interactions between the members of the group, since if the dominant cell is removed, one of the remaining, epidermal, cells will switch fates and become neural. The dominant cell therefore prevents the other cells of the group from becoming neural by a phenomenon known as lateral inhibiton. They, then, adopt the secondary, epidermal, fate. A second class of genes, including the gene shaggy and the neurogenic genes mediate this process. There is some evidence that a proneural cluster is composed of a small number of cells, suggesting a contact-based mechanism of communication. The molecular nature of the protein products of the neurogenic genes is consistent with this idea.     

scabrous Modifies Epithelial Cell Adhesion and Extends the Range of Lateral Signalling during Development of the Spaced Bristle Pattern in Drosophila

The role of scabrous (sca) in the evenly spaced bristle pattern of Drosophila is explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighbouring cells, causing them to adopt the epidermal fate. We show that Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signalling has been increased. scabrous acts nonautonomously, and we present evidence that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signalling are discussed. We also show that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and speculate on a possible role for these structures during signalling.     

Local function of the Notch gene for embryonic ectodermal pathway choice in Drosophila

Mutations at the Notch locus affect the fate of cells in the neurogenic region of the Drosophila embryo so that epidermal precursors become neuroblasts. We have analyzed the cellular requirements for wild-type Notch gene function by means of genetic mosaics, using a cuticle marker to distinguish hypodermal cell genotype. Cells that were genotypically Notch never gave rise to hypoderm within the neurogenic region of mosaic embryos. Mosaic dividing lines within the neurogenic region juxtapose N+ hypoderm with regions of neural hypertrophy. This autonomous action of Notch in hypodermal cells is consistent with a local function of the protein during neurogenesis. Comparison of clone distribution in Notch mosaics and controls suggests that islands of wild-type hypodermal cells fail to differentiate cuticle.     

cis-Inhibition of Notch by Endogenous Delta Biases the Outcome of Lateral Inhibition

Lateral inhibition mediated by Delta/Notch (Dl/N) signaling is used throughout development to limit the number of initially equivalent cells that adopt a particular fate [1], [2] and [3]. Although adjacent cells express both Dl ligand and N receptor, signaling between them ultimately occurs in only one direction. Classically, this has been explained entirely by feedback: activated N can downregulate Dl, amplifying even slight asymmetries in the Dl or N activities of adjacent cells [1], [2], [3], [4] and [5]. Here, however, we present an example of lateral inhibition in which unidirectional signaling depends instead on Dl's ability to inhibit N within the same cell, a phenomenon known as cis-inhibition [6], [7], [8], [9], [10] and [11]. By genetically manipulating individual R1/R6/R7 photoreceptor precursors in the Drosophila eye, we show that loss of Dl-mediated cis-inhibition reverses the direction of lateral signaling. Based on our finding that Dl in R1/R6s requires endocytosis to trans-activate but not to cis-inhibit N, we reexamine previously published data from other examples of lateral inhibition. We conclude that cis-inhibition generally influences the direction of Dl/N signaling and should therefore be included in standard models of lateral inhibition.     

Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function

The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that appears to mediate cell-cell interactions necessary for proper epidermal vs. neural fate decisions. In this study, we examine Notch expression in detail throughout embryonic and imaginal development using confocal laser-scanning microscopy and specific mAb probes. We find that Notch is expressed in a tissue-specific manner as early as the cellular blastoderm stage, when cells of the presumptive mesoderm clearly express less Notch than adjacent ectodermal precursors. Notch is abundantly expressed during the initial determination of neuronal lineages, such as the embryonic neuroblasts and the precursors of sensory neurons in the imaginal disc epithelia, but expression quickly decreases during subsequent differentiation. These changing patterns of Notch expression do not correlate well with cell movements, and thus do not appear to support the notion that the major function of Notch is to maintain epithelial integrity via adhesive mechanisms. Our data suggest instead that Notch may act as a cell-surface receptor, perhaps functioning in the lateral inhibition mechanism that is necessary for proper spacing of neuronal precursors.     

Loss of seven-up from Drosophila R1/R6 photoreceptors reveals a stochastic fate choice that is normally biased by Notch

Recent evidence suggests that stochasticism is important for generating cell type diversity. We have identified a novel stochastic fate choice as part of the mechanism by which Delta/Notch (Dl/N) signaling specifies R7 fate in the Drosophila eye. The equivalence of R1/R6/R7 precursors is normally broken by the activation of N, which specifies the R7 fate. The orphan nuclear hormone receptor Seven-up (Svp) is necessary and sufficient to direct R1/R6/R7 precursors to adopt the R1/R6 fate. A simple model, therefore, is that N represses Svp, which otherwise prevents adoption of the R7 fate. However, we have found that R1/R6s lacking svp stochastically adopt either the R7 or the R8 fate with equal likelihood. We show that N specifies the R7 fate by a novel branched pathway: N represses Svp expression, thereby exposing an underlying stochastic choice between the R7 and R8 fates, and then tips this choice towards the R7 fate.     

Mosaic analysis with oep mutant reveals a repressive interaction between floor-plate and non-floor-plate mutant cells in the zebrafish neural tube

The floor plate is located at the ventral midline of the neural tube in vertebrates. Floor-plate development is severely impaired in zebrafish one-eyed pinhead (oep) mutants. oep encodes a membrane-bound protein with an epiblast growth factor (EGF) motif and functions autonomously in floor-plate precursors. To understand the cell behavior and cell-cell interaction during floor-plate development, the distribution and gene expression of wild-type and oep mutant cells in genetic mosaics were examined. When mutant shield cells were transplanted into a wild-type host, an ectopic neural tube with a floor plate was induced. However, the floor plate of the secondary axis was consistently devoid of mutant cells while its notochord was composed entirely of mutant cells. This indicates that oep shield cells adopt only a notochord fate in a wild-type environment. In reciprocal transplants (wild to oep), however, grafted shield cells frequently contributed to part of the floor-plate region of the secondary neural tube and expressed floor-plate markers. Careful examination of serial sections revealed that a mutant neural cell, when located next to the wild-type cells at the ventral midline, inhibited floor-plate differentiation of the adjacent wild-type cells. This inhibition was effective over an area only one- or two-cells wide along the anteroposterior axis. As the cells located at the ventral midline of the oep neural tube are thought to possess a neural character, similar to those located on either side of the floor plate in a wild-type embryo, this inhibition may play an important role during normal development in restricting the floor-plate region into the ventral-most midline by antagonizing homeogenetic signals from the floor-plate cells.     

Recruitment of cell groups through Delta/Notch signalling during spider neurogenesis

Early neurogenesis in the spider is characterised by a stereotyped pattern of sequential recruitment of neural cells from the neuroectoderm, comparable with neuroblast formation in Drosophila: However, in contrast to Drosophila, where single cells delaminate from the neuroectoderm, groups of cells adopt the neural fate and invaginate into the spider embryo. This raises the question of whether Delta/Notch signalling is involved in this process, as this system normally leads to a singling out of individual cells through lateral inhibition. I have therefore cloned homologues of Delta and Notch from the spider Cupiennius salei and studied their expression and function. The genes are indeed expressed during the formation of neural cells in the ventral neuroectoderm. Loss of function of either gene leads to an upregulation of the proneural genes and an altered morphology of the neuroectoderm that is comparable with Delta and Notch mutant phenotypes in Drosophila: Thus, although Delta/Notch signalling appears to be used in the same way as in Drosophila, the lateral inhibition process produces clusters of invaginating cells, rather than single cells. Intriguingly, neuroectodermal cells that are not invaginating seem to become neural cells at a later stage, while the epidermal cells are derived from lateral regions that overgrow the neuroectoderm. In this respect, the neuroectodermal region of the spider is more similar to the neural plate of vertebrates, than to the neuroectoderm of Drosophila:     

Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors

We have addressed the role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells. We show that mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture showed that a large fraction of Ngn2; Mash1 double-mutant progenitors failed to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate.     

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo

BACKGROUND: In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. RESULTS: To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. CONCLUSIONS: The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.     

A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates

Cells in the neurogenic region of the fly, Drosophila melanogaster, become either neural stem cells or epidermis and the selection of the former requires the activity of the proneural genes [1]. In contrast, it is commonly thought that all cells in the vertebrate neural plate contribute to the neural tube and that consequently there is no need for the selection of individual neural precursors (e.g., [2]). Here we present a detailed fate map of the chick caudal neural plate (CNP), a cell population that generates the posterior hindbrain and spinal cord. We show that this is a unique region of the neural plate where neighbouring cells can contribute to neural tube or epidermis. Further, neural tube precursors leave the CNP in an approximate rostro-caudal order and give rise to discrete portions of the neural tube where they or their progeny behave as neural stem cells [3]. Our data suggest that neural and epidermal cell fates are acquired on a cell-by-cell basis within the CNP and thus in a manner strikingly similar to that in the fly. Indeed, the assignment of neural cell fate in this region may prove to be mediated by the functional homologue of the fly proneural genes (chick achaete-scute homologue 4, cash4), which is expressed heterogeneously within this cell population [4].     

Notch signaling in the nervous system. Pieces still missing from the puzzle

Notch has been known for many years as a receptor for inhibitory signals that shapes the pattern of the nervous system during its development. Genes in the Notch pathway function to prevent neural determination so that only a subset of the available ectodermal cells become neural precursors. The localization of Notch signaling is crucial for determining where neural precursor cells arise on a cell-by-cell basis. The unresolved problem is that studies of the expression of Notch protein and its ligands are inconsistent with the pattern of neurogenesis. During neural cell fate specification, distributions of Notch protein and of its ligand Delta appear uniform. Under the reigning paradigm, such widespread expression should lead to N signal transduction in all cells and thereby prevent any neural specification. Yet, contrary to this expectation, neural elements still form, in characteristic patterns, hence, Notch signal transduction must have been inactive in the precursor cells. The mechanism preventing Notch signaling in certain cells must be posttranslational but it has not yet been identified. This review will outline the experimental evidence supporting this view of Notch signaling, and briefly evaluate some of the possible mechanisms that have been suggested.     

EGF receptor signalling protects smooth-cuticle cells from apoptosis during Drosophila ventral epidermis development

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. We have studied this phenotype and discovered a new function for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning.     

Key role of p63 in BMP-4-Induced Epidermal Commitment of Embryonic Stem Cells

In vivo studies, transgenic and knock-out mice have demonstrated that p63 isoforms play pivotal roles in ectodermal and epidermal development but their respective function remains highly controversial. Since embryonic stem (ES) cells can be differentiated into many cell types, they represent an effective tool to recapitulate in vitro the main steps of embryonic development. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells and clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1+ neural precursors to undergo specific apoptosis while inducing epidermal differentiation through ΔNp63 gene activation. ΔNp63 is not required for ectodermal fate but enhances ES-derived ectodermal cell proliferation and epidermal commitment. This unique cellular model should further provide a powerful tool for identifying the molecular mechanisms controlling normal skin development and in p63-ectodermal dysplasia human congenital pathologies.     

Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling

Graded Hedgehog (Hh) signaling patterns the spinal cord dorsoventral axis by inducing and positioning distinct precursor domains, each of which gives rise to a different type of neuron. These domains also generate glial cells, but the full range of cell types that any one precursor population produces and the mechanisms that diversify cell fate are unknown. By fate mapping and clonal analysis in zebrafish, we show that individual ventral precursor cells that express olig2 can form motoneurons, interneurons and oligodendrocytes. However, olig2+ precursors are not developmentally equivalent, but instead produce subsets of progeny cells in a spatially and temporally biased manner. Using genetic and pharmacological manipulations, we provide evidence that these biases emerge from Hh acting over time to set, maintain, subdivide and enlarge the olig2+ precursor domain and subsequently specify oligodendrocyte development. Our studies show that spatial and temporal differences in Hh signaling within a common population of neural precursors can contribute to cell fate diversification.     

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA-dependent protein kinase PKR

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.