Genetic and Hormonal Regulation of Vitellogenesis in Drosophila

In Drosophila the female-specific yolk protein, or vitellogenin,is synthesized in the fat body. In D. melanogaster, vitellogenin,is first detected in the female hemolymph at the time of adulteclosion and in the ovaries 20 hours later, suggesting differentregulatory mechanisms for the processes of synthesis and uptake.Transplantations of pupal or immature adult ovaries into D.melanogaster adult males induce vitellogenin synthesis, implicatingan ovarian agent in the control of synthesis. Larval ovariesfail to stimulate synthesis. Female-sterile mutants with rudimentaryor previtellogenic ovaries synthesize and accumulate large quantitiesof vitellogenin in the hemolymph, but not in the ovaries. Transplantationof these rudimentary ovaries into males induces vitellogeninsynthesis, suggesting that the ovarian inducing agent does notoriginate from the germ cells. Treatment of the homozygous female-sterilemutants with juvenile hormone stimulates the uptake of vitellogeninby the ovary in some strains. This shows that juvenile hormoneplays a role in vitellogenin uptake. The potential importanceof Drosophila vitellogenesis for studies of gene regulationis discussed.     

Hormonal Interaction in Amphibian Metamorphosis

SYNOPSIS. The climactic stages of amphibian metamorphosis constitutea period characterized by radical morphological changes thatare driven primarily by the thyroidal hormones. Radioimmunoassaysshow that levels of thyroid hormones (TH) rise to a peak duringmetamorphic climax. Accompanying peaks are reported for ACTH,adrenal corticoids (AC), insulin (I) and prolacdn (PRL). ACenhance the metamorphic action of TH by increasing their bindingto nuclei of target cells. TH, in turn, act to raise levelsof AC by stimulating the differentiation of the median eminencethus facilitating the flow of a CRF from the hypothalamus tothe adenohypophysis, by synergizing with ACTH and bystimulationof the interrenals through some other route. During the metamorphicperiod, at least as far as climax, PRL antagonizes TH, perhapsat the thyroidal level and certainly at the level of targetcells. PRL may antagonize by inhibiting induction of hydrolyticenzymes by TH, by alteration of hydromineral responses or byaltering levels of binding of TH to receptors. The antagonisticaction of PRL is mimicked by cAMP. A surge of PRL that is releasedinto the plasma during metamorphic climax seemingly producesno antagonistic effect on thyroidal actions.     

Metamorphosis of the central nervous system of Drosophila

The study of the metamorphosis of the central nervous system of Drosophila focused on the ventral CNS. Many larval neurons are conserved through metamorphosis but they show pronounced remodeling of both central and peripheral processes. In general, transmitter expression appears to be conserved through metamorphosis but there are some examples of possible changes. Large numbers of new, adult-specific neurons are added to this basic complement of persisting larval cells. These cells are produced during larval life by embryonic neuroblasts that had persisted into the larval stage. These new neurons arrest their development soon after their birth but then mature into functional neurons during metamorphosis. Programmed cell death is also important for sculpting the adult CNS. One round of cell death occurs shortly after pupariation and a second one after the emergence of the adult fly.     

Metamorphosis of imaginal discs of Drosophila melanogaster

Summary Imaginal discs ofDrosophila melanogaster larvae, 24–53 hrs after oviposition, were transplanted into mature immobile larval hosts. The transplants did not respond to the hormonal stimuli of metamorphosis, but instead completed their larval development. When reinjected into mature larval hosts, they now differentiated the full set of their presumptive imaginal structures. The process of acquiring competence for metamorphosis appears to be independent of the hormonal conditions.Supported by a credit of the Swiss National Foundation granted to Prof. Dr. E. Hadorn. I thank Dr. R. Nöthiger for his valuable criticism during this investigation.     

Nuclear Melatonin Receptors

Current opinions on the potential role of orphan nuclear retinoid receptors of the ROR/RZR subfamily in regulatory activities of the pineal gland hormone melatonin are reviewed. The mechanisms of receptor–DNA interactions and known coactivators, tissue peculiarities of the expression of different receptor isoforms, and its regulation are described. The spectrum of probable targets for regulation by the receptors, the most promoted of which in this aspect being the immune and central nervous systems, is traced. It is clear that for final adoption of the orphan ROR/RZR receptors, there is need for a full collaboration of endocrinologists for solution of the still debatable questions whether and under which situations melatonin does serve as a physiological modulator of the activities of these receptors.     

Regulation of Drosophila Vasa In Vivo through Paralogous Cullin-RING E3 Ligase Specificity Receptors

In Drosophila species, molecular asymmetries guiding embryonic development are established maternally. Vasa, a DEAD-box RNA helicase, accumulates in the posterior pole plasm, where it is required for embryonic germ cell specification. Maintenance of Vasa at the posterior pole requires the deubiquitinating enzyme Fat facets, which protects Vasa from degradation. Here, we found that Gustavus (Gus) and Fsn, two ubiquitin Cullin-RING E3 ligase specificity receptors, bind to the same motif on Vasa through their paralogous B30.2/SPRY domains. Both Gus and Fsn accumulate in the pole plasm in a Vasa-dependent manner. Posterior Vasa accumulation is precocious in Fsn mutant oocytes; Fsn overexpression reduces ovarian Vasa levels, and embryos from Fsn-overexpressing females form fewer primordial germ cells (PGCs); thus, Fsn destabilizes Vasa. In contrast, endogenous Gus may promote Vasa activity in the pole plasm, as gus females produce embryos with fewer PGCs, and posterior accumulation of Vas is delayed in gus mutant oocytes that also lack one copy of cullin-5. We propose that Fsn- and Gus-containing E3 ligase complexes contribute to establishing a fine-tuned steady state of Vasa ubiquitination that influences the kinetics of posterior Vasa deployment.Establishment and maintenance of polarity is essential for multicellular development. Asymmetric distribution of proteins within cells is often realized by localizing specific mRNAs to distinct positions and by tightly regulating their translation (1). During Drosophila oogenesis, polarized deployment of key mRNAs is crucial for the maternal determination of the embryonic body axes (17). However, although asymmetric mRNA localization within cells is widespread (1, 22), some proteins localize directly. An example is Vasa (Vas), which accumulates in a highly polarized fashion in Drosophila oocytes from a uniformly distributed mRNA that is not believed to be under translational control (8, 20, 21). Vas accumulates in the pole plasm of the oocyte, where it is necessary for embryonic posterior patterning and primordial germ cell (PGC) formation (26). Accumulation of high levels of Vas in the pole plasm requires the deubiquitinating enzyme (DUB) Fat facets (Faf) (25). In faf mutants, levels of posterior Vas are reduced, and polyubiquitinated forms of Vas accumulate. This indicates that Vas stability in the pole plasm is regulated by ubiquitin-dependent pathways.Ubiquitination culminates in the E3 ligase-catalyzed formation of a covalent bond between the C terminus of ubiquitin and a lysine residue of the ubiquitinated protein (12). Target proteins can be ubiquitinated simultaneously and/or sequentially on different lysine residues, and the presence of seven internal lysine residues in ubiquitin itself allows for the formation of topologically distinct polyubiquitin chains (9, 12, 27). Different forms of ubiquitination usually produce different effects on the target protein, and modulation of the steady-state dynamics of ubiquitin conjugation can strongly influence a target''s activity and/or stability. The regulatory logic governing the steady state of target ubiquitination can consist of nonlinear pathways that involve feedback mechanisms, responses to cellular stimuli such as phosphorylation, and complex cross-regulation between individual components of the ubiquitin conjugation machinery and corresponding DUBs.Cullin-RING ubiquitin E3 ligases (CRLs) comprise the largest class of ubiquitin E3 ligases (30). CRLs contain a substrate specificity receptor that binds the ubiquitinated target and a RING protein that is involved in recruiting an E2-conjugating enzyme, which catalyzes transfer of ubiquitin to the associated substrate through the E3 ligase. RING proteins and particular substrate specificity receptors are brought together by scaffold proteins called Cullins, often through small adaptor proteins that link the Cullin with the receptor. Cullin-1 (Cul-1) CRLs recruit their substrate through F-box proteins, with a Skp family adaptor protein forming a bridge between the Cullin and the F-box. In contrast, CRLs containing Cullin-5 (Cul-5) recognize their substrates through receptor proteins that contain a SOCS-box, which are linked to the Cullin by the Elongin B/Elongin C (EloBC) adaptor complex.In this study we identified the F-box protein Fsn and the SOCS-box protein Gus as in vivo regulators of Vas. Fsn and its Caenorhabditis elegans orthologue are required for normal synaptic development and associate with RING proteins encoded by highwire and rpm-1, respectively (24, 46). Gus was previously shown to interact with Vas and was implicated in its posterior localization (37). Gus features a B30.2/SPRY domain through which it directly interacts with a five-amino-acid motif on Vas (DINNN), and it can be cocrystallized with the EloBC complex (44, 45). Fsn has a B30.2/SPRY domain that is very similar to that of Gus (40% identity). We show experimentally that Fsn also binds the DINNN motif and that Gus associates more stably with Vas than does Fsn. Using genetic methods, we investigated the contributions of Gus and Fsn to regulating Vas activity and deployment.     

Nuclear Receptors in Mosquito Vitellogenesis

Vitellogenesis in insects involves the coordinated activityof the fat body, which produces large amounts of yolk proteinprecursors (YP), and oocytes, which specifically accumulatethese proteins. The expression of YP genes is achieved throughstrict sex-, tissue-, and hormone-specific control in the femalefat body. In mosquitoes, expression of YP genes is controlledby 20-hydroxyecdysone (20E). To elucidate the role of 20E inmosquito vitellogenesis, we cloned cDNAs encoding the Aedesaegypti ecdysteroid receptor (AaEcR) and two isoforms of itsheterodimeric partner, the Ultraspiracle homologue (AaUSP).The two AaUSP isoforms differ in their A/B domains and havedistinct expression patterns. The ecdysone regulation of YPgenes likely involves products of early genes. We cloned thegene of the mosquito homologue to the Drosophila early geneE75 (AaE75) belonging to the nuclear receptor superfamily. Kineticsof AaE75 expression correlate with the expression of YP genes,suggesting that AaE75 may have a regulatory role in YP geneexpression. A second nuclear receptor superfamily member, theNGFI-B homologue AaHR38 is implicated in repression of the ecdysone-signalingpathway in the fat body of the previtellogenic female mosquitoat the state-of-arrest. Finally, three isoforms of the hepatocytenuclear factor 4 (HNF-4) homologue AaHNF-4 are differentiallyexpressed in the mosquito fat body during vitellogenesis, suggestingtheir involvement in regulating vitellogenic events in thistissue.