The identification of the Rosa S-locus and implications on the evolution of the Rosaceae gametophytic self-incompatibility systems.

In Rosaceae species, two gametophytic self-incompatibility (GSI) mechanisms are described, the Prunus self-recognition system and the Maleae (Malus/Pyrus/Sorbus) non-self- recognition system. In both systems the pistil component is a S-RNase gene, but from two distinct phylogenetic lineages. The pollen component, always a F-box gene(s), in the case of Prunus is a single gene, and in Maleae there are multiple genes. Previously, the Rosa S-locus was mapped on chromosome 3, and three putative S-RNase genes were identified in the R. chinensis 'Old Blush' genome. Here, we show that these genes do not belong to the S-locus region. Using R. chinensis and R. multiflora genomes and a phylogenetic approach, we identified the S-RNase gene, that belongs to the Prunus S-lineage. Expression patterns support this gene as being the S-pistil. This gene is here also identified in R. moschata, R. arvensis, and R. minutifolia low coverage genomes, allowing the identification of positively selected amino acid sites, and thus, further supporting this gene as the S-RNase. Furthermore, genotype-phenotype association experiments also support this gene as the S-RNase. For the S-pollen GSI component we find evidence for multiple F-box genes, that show the expected expression pattern, and evidence for diversifying selection at the F-box genes within an S-haplotype. Thus, Rosa has a non-self-recognition system, like in Maleae species, despite the S-pistil gene belonging to the Prunus S-RNase lineage. These findings are discussed in the context of the Rosaceae GSI evolution. Knowledge on the Rosa S-locus has practical implications since genes controlling floral and other ornamental traits are in linkage disequilibrium with the S-locus.

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.

Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning.

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.

A role for Ebi in neuronal cell cycle control.

Mutations in ebi were isolated as enhancers of an over-proliferation phenotype generated by elevated E2F/DP activity in the Drosophila eye. ebi alleles also strongly suppress a phenotype caused by the cyclin-dependent kinase inhibitor p21, restoring S phases in the second mitotic wave of the developing eye disk. ebi mutant embryos display ectopic S phases within the peripheral nervous system and central nervous system at a time in development when neuronal precursor cells would normally begin to differentiate. Consistent with this, we find that ebi mutants have a reduced capacity to undergo neuronal differentiation, that Ebi physically interacts with Sina and phyllopod, and that Ebi promotes Ttk88 degradation in vitro and in S2 cells. Ectopic expression of Ttk88 inhibited differentiation in embryos and eye discs; however, this block to differentiation was insufficient to promote S phase entry in either of the situations where ebi mutations gave this effect. We conclude that Ebi has two distinct functions; it promotes the degradation of a repressor of neuronal differentiation (Ttk88), and has a second independent function that limits S phase entry.

Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila.

Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed that crq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria in Drosophila.

Genetic analysis of protein kinase B (AKT) in Drosophila.

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.

u-shaped encodes a zinc finger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila.

The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. The gene u-shaped encodes a novel zinc finger that acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. In contrast, overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects of u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles.

Transcriptional activity of pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila.

The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous alpha globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.

ladybird, a tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis of the Drosophila embryo.

ladybird early and ladybird late genes, tandemly located in the Drosophila 93E homeobox gene cluster, encode highly related homeodomain-containing transcription factors. Here we report the cloning of the complete cDNA sequences of both genes and a study of their expression and regulatory interactions with the segment polarity gene wingless in the epidermis. ladybird genes are co-expressed with wingless in epidermal cells close to the posterior parasegmental boundaries and in terminal regions of the body. In mutant embryos with altered wingless function, transcription of ladybird early and ladybird late is changed; it disappears completely from the epidermis in wingless-embryos, indicating wingless-dependence. After 6 hours of development, wingless expression is maintained by gooseberry in the ventral epidermis. However, in the dorsal epidermis and the terminal regions of the body, expression of wingless is independent of gooseberry but requires a wingless-ladybird regulatory feedback loop. Loss of ladybird function reduces the number of wingless-expressing cells in dorsal epidermis and leads to complete inactivation of wingless in the anal plate. Consequently, mutant ladybird embryos fail to develop anal plates and ubiquitous embryonic expression of either one or both ladybird genes leads to severe defects of the dorsal cuticle. Lack of late wingless expression and anal plate formation can be rescued with the use of a heat-shock-ladybird transgene.

A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila.

A genetic and phenotypic analysis of the gene pannier is described. Animals mutant for strong alleles die as embryos in which the cells of the amnioserosa are prematurely lost. This leads to a dorsal cuticular hole. The dorsal-most cells of the imagos are also affected: viable mutants exhibit a cleft along the dorsal midline. pannier mRNA accumulates specifically in the dorsal-most regions of the embryo and the imaginal discs. Viable mutants and mutant combinations also affect the thoracic and head bristle patterns in a complex fashion. Only those bristles within the area of expression of pannier are affected. A large number of alleles have been studied and reveal that pannier may have opposing effects on the expression of achaete and scute leading to a loss or a gain of bristles.

Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila.

Like the neuroblasts of the central nervous system, sensory organ precursors of the peripheral nervous system of the Drosophila thorax arise as single spaced cells. However, groups of cells initially have neural potential as visualized by the expression of the proneural genes achaete and scute. A class of genes, known as the 'neurogenic genes', function to restrict the proportion of cells that differentiate as sensory organ precursors. They mediate cell communication between the competent cells by means of an inhibitory signal, Delta, that is transduced through the Notch receptor and results in a cessation of achaete-scute activity. Here we show that mutation of either the bHLH-encoding genes of the Enhancer of split complex (E(spl)-C) or groucho, like Notch or Delta mutants, cause an overproduction of sensory organ precursors at the expense of epidermis. The mutant cells behave antonomously suggesting that the corresponding gene products are required for reception of the inhibitory signal. Epistasis experiments place both E(spl)-C bHLH-encoding genes and groucho downstream of Notch and upstream of achaete and scute, consistent with the idea that they are part of the Notch signalling cascade. Since all competent cells produce both the receptor and its ligand, it was postulated that Notch and Delta are linked within each cell by a feedback loop. We show, that, like mutant Notch cells, cells mutant for E(spl)-C bHLH-encoding genes or groucho inhibit neighbouring wild-type cells causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex (AS-C) which must therefore regulate the signal Delta. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(spl)-C and AS-C genes.

pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1.

The gene pannier acts as a repressor of achaete and scute, two transcription factors expressed in discrete subsets of cells at the sites where neural precursors develop. Molecular analysis of mutant alleles revealed the presence of two functional domains within the pannier protein: a zinc finger domain showing homology to the GATA-1 family of vertebrate transcription factors and a domain comprising two putative amphipathic helices. Mutants associated with lesions in the zinc finger domain display an overexpression of achaete and scute and the development of extra neural precursors. Mutant proteins in which the domain including the putative helices is deleted act as hyperactive repressor molecules causing a loss of achaete/scute expression and a loss of neural precursors. Other results suggest that the activity of pannier may be modulated by association with position-specific factors.

Genetic and cytogenetic analysis of the 43A-E region containing the segment polarity gene costa and the cellular polarity genes prickle and spiny-legs in Drosophila melanogaster.

A cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster is presented. Within this interval 27 complementation groups have been identified by extensive F2 screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described.

Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch.

During neurogenesis in Drosophila, groups of equipotential, neurally competent cells choose between epidermal and neural fates. Notch, a phylogenetically conserved transmembrane protein, may act as a receptor in a lateral signalling pathway in which a single neural precursor is chosen from each group and the neural fate of the other cells is inhibited, causing them to differentiate into epidermis. Possible intracellular transduction events mediating signals from Notch are, however, unknown. shaggy is also required for the lateral signal and encodes serine/threonine protein kinases with homology to the glycogen synthase kinase-3 (GSK-3) enzymes that act in signal transduction pathways in vertebrates. We report here that, in transgenic flies, GSK-3 beta can substitute for shaggy, and we also present a study of epistatic relationships between shaggy and gain and loss of function alleles of Notch. The results indicate that shaggy/GSK-3 is part of a signalling pathway downstream of Notch.

Altered epidermal growth factor-like sequences provide evidence for a role of Notch as a receptor in cell fate decisions.

In Drosophila each neural precursor is chosen from a group of cells through cell interactions mediated by Notch and Delta which may function as receptor and ligand (signal), respectively, in a lateral signalling pathway. The cells of a group are equipotential and express both Notch and Delta. Hyperactive mutant Notch molecules, (Abruptex), probably have an enhanced affinity for the ligand. When adjacent to wild-type cells, cells bearing the Abruptex proteins are unable to produce the signal. It is suggested that in addition to the binding of Notch molecules on one cell to the Delta molecules of opposing cells, the Notch and Delta proteins on the surface of the same cell may interact. Binding between a cell's own Notch and Delta molecules would alter the availability of these proteins to interact with their counterparts on adjacent cells.

A dual role for the protein kinase shaggy in the repression of achaete-scute.

achaete and scute are expressed in a spatially restricted pattern and provide neural potential to cells. The domains of expression depend partly on extramacrochaetae whose product is itself spatially restricted and acts as a negative post-translational regulator of achaete and scute. The protein kinase shaggy also represses achaete and scute at many sites but may act via intermediate transcription factors. However shaggy and extramacrochaetae act synergistically and molecular studies suggest that they may be part of the same pathway. shaggy is functionally homologous to the mammalian glycogen synthase kinase-3 and analogy with the known physiology of this enzyme, suggests that this function of shaggy may result from the "constitutive" activity. At the site where a single neural precursor will develop, achaete and scute are initially expressed in a group of equivalent cells. The genes Notch and Delta are part of a lateral signal required to single out one precursor cell and to silence achaete and scute expression in the other cells. shaggy is required downstream of Notch for transduction of the inhibitory signal. This second role of shaggy may be due to modulation of enzymatic activity during signalling.

The choice of cell fate in the epidermis of Drosophila.

In Drosophila, neural precursors are formed in a spaced pattern separated by intervening epidermal cells. Segregation of neural and epidermal lineages relies on cellular interactions. Failure of this cell communication, as in the mutants Notch (N), Delta, and shaggy, results in most or all of the cells becoming neural. Cells mutant for N and shaggy, but not Delta, autonomously adopt the neural fate when adjacent to wild-type cells in mosaics. Furthermore, wild-type cells adopt the epidermal fate if adjacent cells express a lower level of N activity than themselves, but produce neural precursors if adjacent cells express a higher level of N activity. This shows that there is competition between the cells and that the N protein is required for the mechanism whereby the cells choose between alternative fates. It also suggests that N acts as a receptor for an inhibitory signal emanating from the neural precursors.

An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily.

The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster. In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation. Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted. Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function. Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity. Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser. A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles. Ubiquitous expression of sgg transcripts was found during embryonic stages. A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

Mutant Drosophila embryos in which all cells adopt a neural fate.

In the Drosophila embryo, early developmental decisions lead to all cells adopting one of several initial fates, such as those characteristic of the germ layers. The central nervous system is formed subsequently from the neurogenic region of the ectoderm, in which progenitor cells of the neuroblasts and ventral epidermis are intermingled. Two classes of genes govern the segregation of neuroblasts and peripheral sensory organs. The pro-neural class of genes, for example, the achaete-scute complex, participates in the initial decision to make each uniquely positioned neuroblast or sensory organ, but are initially expressed in groups of cells. The segregation of a neuroblast or sensory organ from an equivalent group of equipotential cells involves a mechanism of lateral inhibition whereby the future epidermal cells are prevented from engaging in the primary dominant neural fate. In the absence of this inhibitory signal, all cells of the group will become neural by default. The neurogenic class of genes is thought to mediate these cell interactions. Here we report that cells in embryos mutant for shaggy which are unable to adopt any of the early initial fates, instead develop neural characteristics.