Control of neural precursor specification by proneural proteins in the CNS of Drosophila.

Formation of neural precursors in Drosophila is determined by proneural genes. The distinctive pattern of expression of some genes of the achaete-scute complex in the embryonic neuroectoderm has prompted the speculation that they could also function in the specification of neural precursor identity in the CNS. To test this hypothesis, we have analysed the capacity of different proneural proteins to promote the development of a particular CNS precursor, the MP2 precursor. Our results indicate that: (i) all known proneural proteins are similarly able to support the formation of a neural precursor at the position of MP2; (ii) different proneural proteins promote the expression of different characteristics of MP2; and (iii) a totally normal specification of the MP2 fate can only be attained by the proneural genes achaete or scute.     

Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, we use transgenic Drosophila as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch.     

Embryonic expression of Drosophila ceramide synthase schlank in developing gut, CNS and PNS

Schlank is a member of the highly conserved ceramide synthase family and controls growth and body fat in Drosophila. Ceramide synthases are key enzymes in the sphingolipid de novo synthesis pathway. Ceramide synthase proteins and the (dihydro)ceramide produced are involved in a variety of biological processes among them apoptosis and neurodegeneration. The full extent of their involvement in these processes will require a precise analysis of the distribution and expression pattern of ceramide synthases. Paralogs of the ceramide synthase family have been found in all eukaryotes studied, however the mRNA and protein expression patterns have not yet been analysed systematically. In this study, we use antibodies that specifically recognize Schlank, a schlank mRNA probe and an endogenous schlank promoter driven LacZ reporter line to reveal the expression pattern of Schlank throughout embryogenesis. We found that Schlank is expressed in all embryonic epithelia during embryogenesis including the developing epidermis and the gastrointestinal tract. In addition, Schlank is upregulated in the developing central (CNS) and peripheral nervous system (PNS). Co-staining experiments with neuronal and glial markers revealed specific expression of Schlank in glial and neuronal cells of the CNS and PNS.     

Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

In proneural groups of cells in the morphogenetic furrow of the developing Drosophila eye phosphorylated mitogen activated protein kinase (MAPK) antigen is held in the cytoplasm for hours. We have developed a reagent to detect nuclear MAPK non-antigenically and report our use of this reagent to confirm that MAPK nuclear translocation is regulated by a second mechanism in addition to phosphorylation. This "cytoplasmic hold" of activated MAPK has not been observed in cell culture systems. We also show that MAPK cytoplasmic hold has an essential function in vivo: if it is overcome, developmental patterning in the furrow is disrupted.     

Recruitment of the proneural gene scute to the Drosophila sex-determination pathway

In flies, scute (sc) works with its paralogs in the achaete-scute-complex (ASC) to direct neuronal development. However, in the family Drosophilidae, sc also acquired a role in the primary event of sex determination, X chromosome counting, by becoming an X chromosome signal element (XSE)-an evolutionary step shown here to have occurred after sc diverged from its closest paralog, achaete (ac). Two temperature-sensitive alleles, sc(sisB2) and sc(sisB3), which disrupt only sex determination, were recovered in a powerful F1 genetic selection and used to investigate how sc was recruited to the sex-determination pathway. sc(sisB2) revealed 3' nontranscribed regulatory sequences likely to be involved. The sc(sisB2) lesion abolished XSE activity when combined with mutations engineered in a sequence upstream of all XSEs. In contrast, changes in Sc protein sequence seem not to have been important for recruitment. The observation that the other new allele, sc(sisB3), eliminates the C-terminal half of Sc without affecting neurogenesis and that sc(sisB1), the most XSE-specific allele previously available, is a nonsense mutant, would seem to suggest the opposite, but we show that housefly Sc can substitute for fruit fly Sc in sex determination, despite lacking Drosophilidae-specific conserved residues in its C-terminal half. Lack of synergistic lethality among mutations in sc, twist, and dorsal argue against a proposed role for sc in mesoderm formation that had seemed potentially relevant to sex-pathway recruitment. The screen that yielded new sc alleles also generated autosomal duplications that argue against the textbook view that fruit fly sex signal evolution recruited a set of autosomal signal elements comparable to the XSEs.     

Drosophila tufted is a gain-of-function allele of the proneural gene amos

Tufted is a classical Drosophila mutant characterized by a large number of ectopic mechanosensory bristles on the dorsal mesothorax. Unlike other ectopic bristle mutants, Tufted is epistatic to achaete and scute, the proneural genes that normally control the development of these sensory organs. In this report, I present genetic and molecular evidence that Tufted is a gain-of-function allele of the proneural gene amos that ectopically activates mechanosensory neurogenesis. I also systematically examine the ability of the various proneural bHLH proteins to cross-activate each other and find that their ability to do so is in general relatively limited, despite their common ability to induce the formation of mechanosensory bristles. This phenomenon seems instead to be related to their shared ability to activate Asense and Senseless.     

The abruptex mutations of notch disrupt the establishment of proneural clusters in Drosophila

The receptor encoded by the Notch gene plays a central role in preventing cells from making decisions about their fates until appropriate signals are present. This function of Notch requires the product of the Suppressor of Hairless gene. Loss of either Notch or Suppressor of Hairless function results in cells making premature and incorrect cell fate decisions, whilst increases in Notch signalling prevent cells from making these decisions. Here we find that the proneural clusters are not established correctly in certain Abruptex mutations of Notch and this failure to establish proneural clusters correctly is not due to increased Notch signalling during lateral inhibition. In addition we show that the overexpression of certain dominant negative Notch molecules can disrupt the initiation of proneural cluster development in a manner similar to the Abruptex mutants.     

The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system

In the Drosophila peripheral nervous system, proneural genes direct the formation of different types of sensory organs. Here, we show that amos is a novel proneural gene that promotes multiple dendritic (MD) neuron formation. amos encodes a basic-helix-loop-helix (bHLH) protein of the Atonal family. During embryonic development, amos is expressed in patches of ectodermal cells, and the expression is quickly restricted to sensory organ precursors. Loss of amos function eliminates MD neurons that remain in ASC;atonal mutants. Misexpression of amos generates ectopic MD and other types of neurons. Amos interacts with the ubiquitously expressed Daughter-less protein in vivo and in vitro. Our final misexpression experiments suggest that a domain located outside the DNA-binding domain of Amos determines the MD neuronal specificity.     

Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval PNS. We report a role of scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis we find that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an “off-centred” manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality.     

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.     

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.