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A H Brand

Publications and source records attributed to A H Brand.

At least 19 recordsLinked to original sources
PubMed2000

Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system.

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.

Anaphase↗
PubMed1999

Mastermind acts downstream of notch to specify neuronal cell fates in the Drosophila central nervous system.

In the Drosophila central nervous system, cellular diversity is generated through the asymmetric partitioning of cell fate determinants at cell division. Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. We screened for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. We identified a P-element insertion line that expresses the reporter gene, tau-beta-galactosidase, in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene, mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signalling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. We show that Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signalling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system.

Animals↗
PubMed1998

Miranda mediates asymmetric protein and RNA localization in the developing nervous system.

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.

Animals↗
PubMed1998

Ectopic gene expression in Drosophila using GAL4 system.

Expressing a gene in cells in which it is not normally active is a powerful way of determining its function. The GAL4 system allows the selective expression of any cloned gene in a wide variety of cell- and tissue-specific patterns in Drosophila. A promoter (or enhancer) directs expression of the yeast transcriptional activator GAL4 in a particular pattern, and GAL4 in turn directs transcription of the GAL4-responsive (UAS) target gene in an identical pattern. The system's key feature is that the GAL4 gene and UAS-target gene are initially separated into two distinct transgenic lines. In the GAL4 line, the activator protein is present, but has no target gene to activate. In the UAS-target gene line, the target gene is silent because the activator is absent. It is only when the GAL4 line is crossed to the UAS-target gene line that the target gene is turned on in the progeny. In this article we describe, in detail, how to generate and characterize GAL4 lines and how to prepare UAS-target gene lines. Vector maps are provided for pGaTB, P[GawB], and pP[UAST]. In addition, we consider the range of UAS-reporters currently available and review several new modifications of the GAL4 system.

Animals↗
PubMed1998

Runt determines cell fates in the Drosophila embryonic CNS.

The segmentation gene, runt, is expressed by a subset of the 30 neuroblasts that give rise to each neuromere of the Drosophila embryo. Runt activity in the neuroblasts is necessary for expression of even-skipped in the EL neurons. runt is therefore a good candidate for a gene specifying neuroblast identities. We have ectopically expressed Runt in restricted subsets of neuroblasts and show that Runt is sufficient to activate even-skipped expression in the progeny of specific neuroblasts. Using the marker Tau-green fluorescent protein to highlight the axons, we have found that the extra Even-skipped-expressing neurons project axons along the same pathway as the EL neurons. We find that Runt is expressed in neuroblast 3-3, supporting an autonomous role for runt during neuroblast specification.

Animals↗
PubMed1998

In vivo dynamics of axon pathfinding in the Drosophilia CNS: a time-lapse study of an identified motorneuron.

We developed a system for time-lapse observation of identified neurons in the central nervous system (CNS) of the Drosophila embryo. Using this system, we characterize the dynamics of filopodia and axon growth of the motorneuron RP2 as it navigates anteriorly through the CNS and then laterally along the intersegmental nerve (ISN) into the periphery. We find that both axonal extension and turning occur primarily through the process of filopodial dilation. In addition, we used the GAL4-UAS system to express the fusion protein Tau-GFP in a subset of neurons, allowing us to correlate RP2's patterns of growth with a subset of axons in its environment. In particular, we show that RP2's sharp lateral turn is coincident with the nascent ISN.

Animals↗
PubMed1997

Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila.

Although pioneer neurons are the first to delineate the axon pathways, it is uncertain whether they have unique pathfinding abilities. As a first step in defining the role of pioneer neurons in the Drosophila embryonic CNS, we describe the temporal profile and trajectory of the axons of four pioneer neurons and show that they differ from previously published reports. We show, by targeted ablation of one, two, three or four pioneer neurons at a time, that (1) no single pioneer neuron is essential for axon tract formation, (2) the interaction between two pioneers is necessary for the establishment of each fascicle and (3) pioneer neurons function synergistically to establish the longitudinal axon tracts, to guide the fasciculation of follower neurons along specific fascicles and to prevent axons from crossing the midline.

Animals↗
PubMed1996

Paclitaxel (Taxol) as salvage therapy for relapsed ovarian cancer.

Thirty-two patients with epithelial ovarian cancer received paclitaxel 175 mg/m2, by 3-hour infusion, in this prospective phase 2 study. All patients had relapsed or progressed after initial cisplatin/cyclophosphamide therapy. Thirteen patients received paclitaxel as second line therapy, 14 as third line therapy and 5 as fourth line therapy. One patient was excluded from efficacy analysis, due to a severe anaphylactic reaction to the first cycle of paclitaxel therapy. Of the 31 evaluable patients, complete remission was observed in 3 patients (9.7%) and partial remission in 11 patients (35.5%), with a total response rate of 45.2%. The median survival from diagnosis for the 31 evaluable patients was 32.5 months and the median survival following therapy with paclitaxel was 12.2 months (range 4-27 months). The 3 patients who achieved a complete response remain alive, at greater than 20 months, since commencing paclitaxel. Twelve patients (38.7%) who achieved a partial response, or had stable disease, had a median survival, after paclitaxel treatment, of greater than 18 months. This study confirms the activity of paclitaxel in relapsed ovarian cancer and demonstrates a prolonged survival benefit in greater than one third of this group of patients.

Adult↗
PubMed1995

Evidence for engrailed-independent wingless autoregulation in Drosophila.

Proper spatial expression of the wingless (wg) gene in the Drosophila embryonic epidermis is crucial to intrasegmental patterning. Single cell wide wg expression is initiated at the blastoderm stage in response to combinatorial regulation by the pair rule genes. Later, during gastrulation, when the epidermal expression of the pair rule genes has disappeared, wg becomes regulated by the activity of the segment polarity genes. The segment polarity gene engrailed (en) is expressed in cells adjacent to the wg-expressing cells and is required to maintain wg transcription. Since wg is in turn required to maintain en expression, wg appears to autoregulate its own expression through an endependent paracrine feedback loop. In this paper, we demonstrate that wild-type wg expression requires wg activity during stage 9, prior to its requirement for en maintenance, indicating that wg has an autoregulatory role that is distinct from its paracrine feedback loop through en. In addition, by misexpressing Wg and En in distinct spatial patterns in the epidermis, we find that En is capable of inducing expression from the endogenous wg gene only in immediate adjacent cells which have been exposed to Wg. Furthermore, exogenous Wg expression enables maintenance of endogenous wg transcription in both wg and en mutant embryos. Our results support the model that in the wild-type embryo, wg has an autoregulatory function which is distinct and separable from paracrine regulation via en. We also provide evidence that late, localized Wg expression is crucial for the asymmetric patterning of epidermal cell types as reflected in the larval cuticle.

Animals↗
PubMed1995

Grapelike leiomyoma of the uterus.

A 24-year-old nulliparous woman underwent laparotomy for a large pelvic mass. Grapelike tumor extending from the uterus into the broad ligaments and peritoneal cavity was found. A diagnosis of sarcoma appeared likely, but radical surgery was avoided when frozen sections indicated a histologically benign smooth muscle tumor.

Adult↗
PubMed1995

The GAL4 system as a tool for unravelling the mysteries of the Drosophila nervous system.

The GAL4 system is a method for directed gene expression that allows genes to be expressed ectopically in numerous cell- or tissue-specific patterns. The technique is being exploited to study the Drosophila melanogaster nervous system at all stages of development, from the embryo to the adult. The GAL4 system is being used to target the expression of novel marker genes in living animals to label cells, or subcellular structures. Directed expression of toxin genes can be used as a method for targeted cell ablation to study the role of cell-cell interactions in development. Ectopic expression helps to elucidate the function of different genes in cell fate determination and differentiation, and is helping to define the regions of the brain involved in sexual behaviour.

Animals↗
PubMed1995

Targeted ablation of glia disrupts axon tract formation in the Drosophila CNS.

Glial cells are thought to play a role in growth cone guidance, both in insects and in vertebrates. In the developing central nervous system of the Drosophila embryo, the interface glia form a scaffold prior to the extension of the first pioneer growth cones. Growing axons appear to contact the glial scaffold as the axon tracts are established. We have used a novel technique for targeted cell ablation to kill the interface glia and thus to test their role in establishment of the embryonic axon tracts. We show that ablation of the interface glia early in development leads to a complete loss of the longitudinal axon tracts. Ablation of the glia later in embryonic development results in defects comprising weakening and loss of axon fascicles within the connectives. We conclude that the interface glia are required first for growth cone guidance in the formation of the longitudinal axon tracts in the Drosophila embryo and then either to direct the follower growth cones, or to maintain the longitudinal axon tracts.

Animals↗
PubMed1994

Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis.

In Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. We describe a new role for Raf in Drosophila development, showing that Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. We show that Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, in that either can induce ventral follicle cells to assume a dorsal fate.

Animals↗
PubMed1994

Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A.

Reported assays of the bone morphogenetic proteins (BMPs) have not in general revealed specific functions for the different proteins, belying the specificity implied by the evolutionary conservation and distinct expression patterns of the genes encoding BMPs. We have used assays of developmental function to show that the two Drosophila homologues of the BMPs, decapentaplegic (dpp) and 60A, that both induce ectopic bone formation in mammalian assay systems, have distinct effects in Drosophila development. A binary expression system using the yeast transcriptional activator GAL4 directed identical patterns of tissue and temporally specific dpp and 60A expression. When dpp enhancer elements drove GAL4 expression, GAL4-responsive dpp transgenes rescued dpp mutant phenotypes, but GAL4-responsive 60A transgenes did not. Ectopic ectodermal expression of dpp during gastrulation respecified the dorsal/ventral pattern of the embryo. In contrast, ectopic 60A expression had no detectable effects on embryonic development but led to defects in adult structures or lethality during metamorphosis. Expression of 60A in cells expressing dpp did not interfere with dpp functions, indicating that dysfunctional heterodimers did not form at sufficient levels to inhibit dpp. These specific developmental responses in Drosophila indicate that in vivo functions of BMP-like factors can be more specific than indicated by the ectopic bone formation assays and that the Drosophila embryo provides an assay system sensitive to the structural differences that contribute to BMP specificity in vivo.

Amino Acid Sequence↗
PubMed1993

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Animals↗
PubMed1989

RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML.

DNA fragments containing the silencers that flank the mating type genes at HML alpha are shown to bind specifically to the nuclear scaffold of yeast. The scaffold proteins are solubilized with urea and then renatured to form a soluble extract which allows reconstitution of sequence-specific DNA loops. At the silent mating type locus HML alpha, loops are formed by either silencer-silencer (E-I) interaction or silencer-promoter interactions (E-P and I-P). The nuclear protein RAP-1 fractionates efficiently with the nuclear scaffold, and binds to the E, I, and promoter regions. Affinity purification of RAP-1 and oligonucleotide competition show that RAP-1 is necessary for reconstitution of loops in vitro. These results are consistent with a model in which silencers define a chromatin loop within which occur modifications that maintain the promoter in an inactive state.

Chromosome Mapping↗