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.     

Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway.     

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.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

An EP overexpression screen for genetic modifiers of Notch pathway function in Drosophila melanogaster

The Notch pathway comprises a signal transduction cascade required for the proper formation of multiple tissues during metazoan development. Originally described in Drosophila for its role in nervous system formation, the pathway has attracted much wider interest owing to its fundamental roles in a range of developmental and disease-related processes. Despite extensive analysis, Notch signaling is not completely understood and it appears that additional components of the pathway remain to be identified and characterized. Here, we describe a novel genetic strategy to screen for additional Notch pathway genes. The strategy combines partial loss of function for pathway activity with Enhancer-promoter (EP)-induced overexpression of random loci across the dorsoventral wing margin. Mastermind (Mam) is a nuclear component of the Notch signaling cascade. Using a GAL4-UAS-driven dominant-negative form of Mam, we created a genotype that exhibits a completely penetrant dominant wing-nicking phenotype. This phenotype was assayed for enhancement or suppression after outcrossing to several thousand EP lines. The screen identified known components or modifiers of Notch pathway function, as well as several potential new components. Our results suggest that a genetic screen that combines partial loss of function with random gene overexpression might be a useful strategy in the analysis of developmental pathways.     

DRR1 is expressed in the developing nervous system and downregulated during neuroblastoma carcinogenesis

Down-regulated in renal cell carcinoma 1 (DRR1) is mapped at 3p21.1, and is a candidate tumor suppressor gene. However, its biological roles have yet to be elucidated. Here, we developed polyclonal antibodies against DRR1 protein, and examined its expression during embryogenesis and carcinogenesis. The DRR1 protein was preferentially expressed in axonal projections of the central and peripheral nervous system of mice during embryonic days 10.5-16.5. Consistent with this expression pattern, the protein was detected in the neurites of primary cultured cortical neurons of rats at embryonic day 18.5. Survival of these cells was significantly inhibited by RNAi-induced downregulation of DRR1 expression. DRR1 was poorly expressed in established cancer cell lines, including neuroblastoma cells, whereas strong expression was observed in normal cells. A neuroblastoma model, MYCN transgenic mice, revealed that DRR1 protein was expressed in the celiac ganglion 2 weeks after birth when neuroblast hyperplasia was also observed; however, there was no longer any expression of DRR1 protein in tumors originating from the ganglion 8 weeks after birth. Together, our data indicate that DRR1 protein is expressed in normal cells, particularly in the nervous system during embryogenesis, is involved in neuronal cell survival, and is downregulated during neuroblastoma carcinogenesis.     

Shh signal and its functional roles in normal and diseased brain

The identification of a Sonic Hedgehog (Shh) signaling pathway in the adult vertebrate central nervous system has paved the way to the characterization of the functional roles of Shh signals in normal and diseased brain. This morphogen is proposed to play a key role in the establishment and maintenance of adult neurogenic niches and to modulate the proliferation of neuronal or glial precursors. Consistent with its role during embryogenesis, alteration of Shh signaling is associated with tumorigenesis while its recruitment in damaged neural tissue might be part of the regenerating process. We will discuss the most recent data of the Hedgehog pathway in the adult brain and its relevance as a novel therapeutic approach for brain diseases including brain tumors.     

Early ventral expression of the Drosophila neurogenic locus mastermind

The neurogenic locus mastermind (mam) of Drosophila is required for the segregation of epidermal from neural cell lineages. Previous studies have shown that during neurogenesis mam appears to be expressed throughout the ectoderm, mesoderm, and neuroblast layer of the germ band. Here it is demonstrated that during early embryogenesis mam is expressed ubiquitously; however, the predominant domains of accumulation of mam RNA and protein during gastrulation are along the ventral longitudinal surface, including cells of the mesoderm, endoderm, mesectoderm, and neuroectoderm. The regions of elevated mam accumulation coincide with the realm of action of dorsoventral patterning genes.     

Alternative protein secretion: the Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast

To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.     

Restriction of neurogenic ability during neural crest cell differentiation

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells. © 1993 John Wiley & Sons, Inc.     

Regulation of Drosophila neural development by a putative secreted protein

The Drosophila strawberry (sty) locus was isolated by P-element insertion mutagenesis in a screen for mutations affecting eye development. Analysis of the mutant phenotype and the putative expression pattern of the sty gene suggested that it has multiple functions. Mutations in the sty gene lead to irregular spacing of ommatidia, an increase in the number of photoreceptor cells, as well as abnormal axonal projections to the lamina and disrupted structure of the optic lobes in the adult fly. The sty mutation also causes abnormal head involution, a change in a number of sensilla in the antennomaxillary complex in the embryonic stage and abnormal morphogenesis of the maxillary palp and wings in later stages. We examined the presumptive expression of the sty gene during development by histochemical staining for lacZ expression from enhancer trap elements inserted within the sty gene. During embryogenesis, expression of lacZ showed a segmental pattern in the ectoderm and in the nervous system. In the eye imaginal discs, lacZ was expressed in photoreceptor cells beginning a few rows posterior to the morphogenetic furrow. The lacZ was also expressed in the wing disc. In the adult, lacZ was expressed in the retina and lamina. We cloned the sty gene by P-element tagging and found that it encodes a putative secreted protein containing a cysteine-rich region similar to the epidermal growth factor (EGF) repeat. On the basis of the loss of functional phenotype, the expression pattern and the predicted structure of its product, we propose that sty encodes a diffusible protein acting as a signal involved in lateral inhibition within the developing nervous system and also as a factor involved either directly or indirectly in axonal guidance and optic lobe development.