Graded requirement for the zygotic terminal gene, tailless, in the brain and tail region of the Drosophila embryo

We have used hypomorphic and null tailless (tll) alleles to carry out a detailed analysis of the effects of the lack of tll gene activity on anterior and posterior regions of the embryo. The arrangement of tll alleles into a continuous series clarifies the relationship between the anterior and posterior functions of the tll gene and indicates that there is a graded sensitivity of anterior and posterior structures to a decrease in tll gene activity. With the deletion of both anterior and posterior pattern domains in tll null embryos, there is a poleward expansion of the remaining pattern. Using anti-horseradish peroxidase staining, we show that the formation of the embryonic brain requires tll. A phenotypic and genetic study of other pattern mutants places the tll gene within the hierarchy of maternal and zygotic genes required for the formation of the normal body pattern. Analysis of mutants doubly deficient in tll and maternal terminal genes is consistent with the idea that these genes act together in a common pathway to establish the domains at opposite ends of the embryo. We propose that tll establishes anterior and posterior subdomains (acron and tail regions, respectively) within the larger pattern regions affected by the maternal terminal genes

Drosophila arc Encodes a Novel Adherens Junction-Associated PDZ Domain Protein Required for Wing and Eye Development

AbstractLoss of arc function results in a downwardly curved wing and smaller eyes with a reduced number of ommatidia. Consistent with this phenotype, molecular analysis shows that arc mRNA and protein are expressed in the wing imaginal disc and in clusters of cells in the morphogenetic furrow of the eye imaginal disc. The 36-kb arc transcription unit contains 10 exons that are spliced to form a 5.5-kb mRNA. The encoded Arc protein is 143,000 Da and contains two PDZ (PSD-95, Discs large, ZO-1) domains; there is no close structural similarity to other PDZ proteins. In addition to its expression in imaginal discs, arc is expressed during embryogenesis in epithelia undergoing morphogenesis, including the invaginating posterior midgut, evaginating Malpighian tubule buds, elongating hindgut, invaginating salivary glands, intersegmental grooves, and developing tracheae. Arc protein colocalizes with Armadillo (β-catenin) to the apical (luminal) surface of these developing epithelia, indicating that it is associated with adherens junctions. Genes that are required for patterning of embryonic epithelia (e.g., tailless, Krüppel, fork head, and brachyenteron) or for progression of the morphogenetic furrow (i. e., hedgehog) are required to establish or maintain the regional expression of arc. Misexpression of arc in the eye imaginal discs results in rough and larger eyes with fused ommatidia. We propose that arc affects eye development by modulating adherens junctions of the developing ommatidium

A Drosophila Neurexin Is Required for Septate Junction and Blood-Nerve Barrier Formation and Function

AbstractSeptate and tight junctions are thought to seal neighboring cells together and to function as barriers between epithelial cells. We have characterized a novel member of the neurexin family, Neurexin IV (NRX), which is localized to septate junctions (SJs) of epithelial and glial cells. NRX is a transmembrane protein with a cytoplasmic domain homologous to glycophorin C, a protein required for anchoring protein 4.1 in the red blood cell. Absence of NRX results in mislocalization of Coracle, a Drosophila protein 4.1 homolog, at SJs and causes dorsal closure defects similar to those observed in coracle mutants. nrx mutant embryos are paralyzed, and electrophysiological studies indicate that the lack of NRX in glial–glial SJs causes a breakdown of the blood-brain barrier. Electron microscopy demonstrates that nrx mutants lack the ladder-like intercellular septa characteristic of pleated SJs (pSJs). These studies identify NRX as the first transmembrane protein of SJ and demonstrate a requirement for NRX in the formation of septate-junction septa and intercellular barriers

The Drumstick/Lines/Bowl regulatory pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal cell differentiation

Hedgehog and Wingless signaling in the Drosophila embryonic epidermis represents one paradigm for organizer function. In patterning this epidermis, Hedgehog and Wingless act asymmetrically, and consequently otherwise equivalent cells on either side of the organizer follow distinct developmental fates. To better understand the downstream mechanisms involved, we have investigated mutations that disrupt dorsal epidermal pattern. We have previously demonstrated that the gene lines contributes to this process. Here we show that the Lines protein interacts functionally with the zinc-finger proteins Drumstick (Drm) and Bowl. Competitive protein-protein interactions between Lines and Bowl and between Drm and Lines regulate the steady-state accumulation of Bowl, the downstream effector of this pathway. Lines binds directly to Bowl and decreases Bowl abundance. Conversely, Drm allows Bowl accumulation in drm-expressing cells by inhibiting Lines. This is accomplished both by outcompeting Bowl in binding to Lines and by redistributing Lines to the cytoplasm, thereby segregating Lines away from nuclearly localized Bowl. Hedgehog and Wingless affect these functional interactions by regulating drm expression. Hedgehog promotes Bowl protein accumulation by promoting drm expression, while Wingless inhibits Bowl accumulation by repressing drm expression anterior to the source of Hedgehog production. Thus, Drm, Lines, and Bowl are components of a molecular regulatory pathway that links antagonistic and asymmetric Hedgehog and Wingless signaling inputs to epidermal cell differentiation. Finally, we show that Drm and Lines also regulate Bowl accumulation and consequent patterning in the epithelia of the foregut, hindgut, and imaginal discs. Thus, in all these developmental contexts, including the embryonic epidermis, the novel molecular regulatory pathway defined here is deployed in order to elaborate pattern across a field of cells

Decreased GH secretion and enhanced ACTH and cortisol release after ghrelin administration in Cushing's disease: comparison with GHRP-6

GHRP-6-induced GH release is decreased in Cushing's disease (CD), and ACTH and cortisol responses are enhanced. Ghrelin stimulates GH, ACTH and cortisol release in men. We compared the GH, ACTH and cortisol responses to ghrelin and GHRP-6 in CD (n = 12) and controls (n = 9). In CD, peak GH levels (mu g/L; mean +/- SE) after ghrelin administration (7,7 +/- 2,6) were higher than those observed after GHRP-6 (2,7 +/- 1), but lower than in controls (58,4 +/- 10). Peak ACTH (pg/mL) and cortisol (mu g/dL) levels after ghrelin (187 +/- 35 +/- 4,5) were higher than in controls (93 +/- 32; 18,9 +/- 1,1), but similar to GHRP-6 (185 +/- 57; 33,3 +/- 4,7). The higher GH response to ghrelin compared to GHRP-6 in CD suggests activation of additional intracellular pathways by ghrelin. The same ACTH and cortisol releasing mechanisms are probably stimulated by both peptides.Univ Fed Sao Paulo, Div Endocrinol, EPM, FAPESP,CNPq, Sao Paulo, BrazilUniv Fed Sao Paulo, Div Endocrinol, EPM, FAPESP,CNPq, Sao Paulo, BrazilWeb of Scienc

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors.

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an 'upper' and 'lower' moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure-activity relationships, and empower future structure-based drug design campaigns.F. Hoffmann-La Roche Lt

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors.

Acknowledgements: We acknowledge L. Cooper for training in cryo-EM grid preparation and for performing grid clipping; J. Stayaert (Vrije Universiteit Brussel) for kindly providing MbF3; M. Reutlinger and J. Benz for scientific discussions early in the project; V. Graf for electrophysiology support; and M. Karg, M. Fogetta and M. Siegrist for molecular biology support. This work was supported by a BBSRC project grant, BB/M024709/1 (P.S.M.), the Department of Pharmacology new lab start-up fund, and the University of Cambridge Isaac Newton & Wellcome Trust Institutional Strategic Support Fund, Academy of Medical Sciences Springboard Award, SBF004\1074 (P.S.M.), and funding from F. Hoffmann-La Roche Ltd. The cryo-EM facility receives funding from the Wellcome Trust, 206171/Z/17/Z; 202905/Z/16/Z (S.W.H. and D.Y.C.) and University of Cambridge.α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an 'upper' and 'lower' moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure-activity relationships, and empower future structure-based drug design campaigns.F. Hoffmann-La Roche Lt