Daughterless coordinates somatic cell proliferation,differentiation and germline cyst survival during follicle formation in Drosophila

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, we have identified several specific requirements for da(+) in somatic cells during follicle formation. First, da is a critical regulator of somatic cell proliferation. Also, da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. Finally, da is a genetic regulator of an early checkpoint for germline cyst progression: Loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles.     

Chronic cocaine exposure in Drosophila: life, cell death and oogenesis

Developmental signaling cascades that can be perturbed by cocaine and other drugs of abuse have been difficult to study in humans and vertebrate models. Although numerous direct neural targets of cocaine have been elucidated at the molecular level, little is known about the specific cellular events that are impacted indirectly as a result of the drug's perturbation of neural circuits. We have developed oogenesis in Drosophila melanogaster as a model in which to identify downstream biochemical and/or cellular processes that are disrupted by chronic cocaine exposure. In this model, cocaine feeding resulted not only in expected reductions in viability, but also in unanticipated developmental defects during oogenesis, including aberrant follicle morphogenesis and vitellogenic follicle degeneration. To identify mechanisms through which cocaine exerted its deleterious effects on oogenesis, we examined candidate components of neural and hormonal signaling pathways. Cocaine-induced disruptions in follicle formation were enhanced by juvenile hormone exposure and phenocopied by serotonin feeding, while cocaine-activated follicle apoptosis was enhanced by concomitant dopamine feeding. HPLC analysis of dopamine and serotonin in the ovary suggests that these neurotransmitters could variably mediate cocaine's effects on oogenesis indirectly in the brain and/or directly in the ovary itself. We confirmed the involvement of hormone signaling by measuring ecdysteroids, which increase following cocaine exposure, and by demonstrating suppression of cocaine-induced follicle loss by hormone receptor mutants. Cocaine-induced ovarian follicle apoptosis and adult lethality appear to be caused by modulation of dopamine levels, while morphological defects during follicle formation likely result from perturbing serotonin signaling during cocaine exposure. Our work suggests not only a new role for juvenile hormone and/or serotonin in Drosophila ovarian follicle formation, but also a cocaine-sensitive role for dopamine in modulating hormone levels in the female fly.     

Maternal phosphatase inhibitor-2 is required for proper chromosome segregation and mitotic synchrony during Drosophila embryogenesis

Protein phosphatase-1 (PP1) is a major Ser/Thr phosphatase conserved among all eukaryotes, present as the essential GLC7 gene in yeast. Inhibitor-2 (I-2) is an ancient PP1 regulator, named GLC8 in yeast, but its in vivo function is unknown. Unlike mammals with multiple I-2 genes, in Drosophila there is a single I-2 gene, and here we describe its maternally derived expression and required function during embryogenesis. During oogenesis, germline expression of I-2 results in the accumulation of RNA and abundant protein in unfertilized eggs; in embryos, the endogenous I-2 protein concentrates around condensed chromosomes during mitosis and also surrounds interphase nuclei. An I-2 loss-of-function genotype is associated with a maternal-effect phenotype that results in drastically reduced progeny viability, as measured by reduced embryonic hatch rates and larval lethality. Embryos derived from I-2 mutant mothers show faulty chromosome segregation and loss of mitotic synchrony in cleavage-stage embryos, patchy loss of nuclei in syncytial blastoderms, and cuticular pattern defects in late-stage embryos. Transgenic expression of wild-type I-2 in mutant mothers gives dose-dependent rescue of the maternal effect on embryo hatch rate. We propose that I-2 is required for proper chromosome segregation during Drosophila embryogenesis through the coordinated regulation of PP1 and Aurora B.     

Modification of chitosan-bead support materials with l-lysine and l-asparagine for α-amylase immobilization

Bioprocess and Biosystems Engineering - Maltose syrups have got wide-range utilizations in a variety of applications from bakery to drug-development. α-Amylases are among the most widely...     

The Drosophila sex determination gene daughterless has different functions in the germ line versus the soma

As a regulator of the female-specific gene Sxl, da+ provides an essential maternal component in the control of sex determination and dosage compensation; nevertheless, neither the maternal nor zygotic phenotypes of the original mutant da allele is sex-specific. Here we clarify the role of da+ in Drosophila development, finding: this sex determination gene is indeed pleiotropic; zygotic functioning of da+ is essential in both sexes for somatic cell development, but not for germ cell development; da female sterility results from a somatic, rather than germ-line, defect; and expression of da+ in the maternal germ line is required only for daughters in the subsequent generation, as expected for a specific regulator of Sxl+. These conclusions follow from the characterization of new da null alleles isolated by a selection for defects in maternally acting positive regulators of Sxl.     

stall-mediated extrinsic control of ovarian follicle formation in Drosophila

Complex patterns of morphogenesis require intricate coordination of multiple, regulatory processes that control cellular identities, shapes, and behaviors, both locally and over vast distances in the developing organism or tissue. Studying Drosophila oogenesis as a model for tissue morphogenesis, we have discovered extraovarian regulation of follicle formation. Clonal analysis and ovary transplantation have demonstrated that long-range control of follicle individualization requires stall gene function in cells outside of the ovary. Although tissue nonautonomous regulation has been shown to govern follicle maturation and survival, this is the first report of an extraovarian pathway involved in normal follicle formation.     

Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Type II cAMP-dependent protein kinase-deficient Drosophila are viable but show developmental, circadian, and drug response phenotypes

We identified a unique type II cAMP-dependent protein kinase regulatory subunit (PKA-RII) gene in Drosophila melanogaster and a severely hypomorphic if not null mutation, pka-RII(EP(2)2162). Extracts from pka- RII(EP(2)2162) flies selectively lack RII-specific autophosphorylation activity and show significantly reduced cAMP binding activity, attributable to the loss of functional PKA-RII. pka-RII(EP(2)2162) shows 2-fold increased basal PKA activity and approximately 40% of normal cAMP-inducible PKA activity. pka-RII(EP(2)2162) is fully viable but displays abnormalities of ovarian development and multiple behavioral phenotypes including arrhythmic circadian locomotor activity, decreased sensitivity to ethanol and cocaine, and a lack of sensitization to repeated cocaine exposures. These findings implicate type II PKA activity in these processes in Drosophila and imply a common role for PKA signaling in regulating responsiveness to cocaine and alcohol.