Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification

The Drosophila compound eye is specified by the concerted action of seven nuclear factors that include Eyeless/Pax6. These factors have been called "master control" proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, we find that several of these eye specification genes are not coexpressed in the same embryonic cells-or even in the presumptive eye. We demonstrate that the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes, and show that specification occurs much later than previously thought-not during embryonic development but in the second larval stage.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

The coiled-coil protein shrub controls neuronal morphogenesis in Drosophila

The diversity of neuronal cells, especially in the size and shape of their dendritic and axonal arborizations, is a striking feature of the mature nervous system. Dendritic branching is a complex process, and the underlying signaling mechanisms remain to be further defined at the mechanistic level. Here we report the identification of shrub mutations that increased dendritic branching. Single-cell clones of shrub mutant dendritic arborization (DA) sensory neurons in Drosophila larvae showed ectopic dendritic and axonal branching, indicating a cell-autonomous function for shrub in neuronal morphogenesis. shrub encodes an evolutionarily conserved coiled-coil protein homologous to the yeast protein Snf7, a key component in the ESCRT-III (endosomal sorting complex required for transport) complex that is involved in the formation of endosomal compartments known as multivesicular bodies (MVBs). We found that mouse orthologs could substitute for Shrub in mutant Drosophila embryos and that loss of Shrub function caused abnormal distribution of several early or late endosomal markers in DA sensory neurons. Our findings demonstrate that the novel coiled-coil protein Shrub functions in the endosomal pathway and plays an essential role in neuronal morphogenesis.     

Regulatory roles of presenilin-1 and nicastrin in neuronal differentiation during in vitro neurogenesis

Presenilin (PS) in association with nicastrin (NICA) forms a gamma-secretase complex that plays a crucial role in facilitating intramembranous processing of Notch, a signaling receptor that is essential for neuronal fate specification and differentiation. Loss of function studies have implicated a role for PS1 in regulating neuronal differentiation in association with the down-regulation of Notch signaling during neurogenesis. By using a system for stable, as well as tetracycline-inducible expression of interfering RNAs (RNAi), we studied the functions of PS1 during neuronal differentiation in the murine pluripotent p19 embryonic carcinoma cell line. After retinoic acid (RA) treatment and in the absence of doxycycline, neuronal progenitor cells in the p19 clone were found to extend their processes towards the neighboring colony to form network-like connections, as revealed by neuron-specific microtubule-associated protein 2 staining and laser scanning confocal microscopy. However, doxycycline-induced expression of PS1 small interfering RNA (siRNA) in the p19 clone resulted in a severe defect in the formation of network-like connections. Expression of the NICA and Notch down-stream effector genes Hes1 and Hes5 was unaffected in p19 cells expressing doxycycline-induced PS1 siRNA. In contrast to PS1, constitutive inactivation of NICA by siRNA in p19 cells resulted in premature and partial differentiation without RA treatment. In these NICA siRNA-expressing p19 cells the expression of the Notch1 down-stream effector Hes1 gene was substantially reduced. After RA treatment the NICA siRNA clone failed to differentiate completely into networks of neurons. These results taken together provide direct evidence that PS1 and NICA may participate in neuronal differentiation during neurogenesis in vitro.     

Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors

The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.     

Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding

Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect beta-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that beta-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, beta-Spectrin is associated with alpha-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of beta-Spectrin significantly reduces levels of neuronal alpha-Spectrin expression, whereas gain of beta-Spectrin leads to an increase in alpha-Spectrin protein expression. Because the loss of alpha-spectrin does not result in an embryonic nervous system phenotype, beta-Spectrin appears to act at least partially independent of alpha-Spectrin to control axonal patterning.     

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile X-related (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect.     

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.     

OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development

Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.