Gretchen Calhoun

Novel Functions of nanos in Downregulating Mitosis and Transcription during the Development of the Drosophila Germline

It has previously been shown that germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several germline-specific markers. In the studies reported here, we have traced these defects back to the syncytial blastoderm stage. We show that pole cells in nos embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped are ectopically activated in nos- germ cells. We show that nos- germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos- germ cells. Supporting the conclusion that Sxl is an important target for nos repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity.

The JAK/STAT Signaling Pathway Is Required for the Initial Choice of Sexual Identity in Drosophila melanogaster

The choice of sexual identity in Drosophila is determined by a system that measures the X chromosome to autosome ratio (X/A). This system depends upon unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sxl promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, we generated a gain-of-function promoter, Sxl-PeGOF, that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. We demonstrate that the JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation.

AN N-TERMINAL TRUNCATION UNCOUPLES THE SEX-TRANSFORMING AND DOSAGE COMPENSATION FUNCTIONS OF SEX-LETHAL

ABSTRACT In Drosophila melanogaster, Sex-lethal(Sxl) controls autoregulation and sexual differentiation by alternative splicing but regulates dosage compensation by translational repression. To elucidate how Sxl functions in splicing and translational regulation, we have ectopically expressed a full-length Sxl protein (Sx.FL) and a protein lacking the N-terminal 40 amino acids (Sx-N). The Sx.FL protein recapitulates the activity ofSxl gain-of-function mutations, as it is both sex transforming and lethal in males. In contrast, the Sx-N protein unlinks the sex-transforming and male-lethal effects of Sxl. The Sx-N proteins are compromised in splicing functions required for sexual differentiation, displaying only partial autoregulatory activity and almost no sex-transforming activity. On the other hand, the Sx-N protein does retain substantial dosage compensation function and kills males almost as effectively as the Sx.FL protein. In the course of our analysis of the Sx.FL and Sx-N transgenes, we have also uncovered a novel, negative autoregulatory activity, in which Sxl proteins bind to the 3′ untranslated region of Sxl mRNAs and decrease Sxl protein expression. This negative autoregulatory activity may be a homeostasis mechanism.

Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline

In Drosophila melanogaster, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, we ectopically expressed a potent transcriptional activator, Bicoid (Bcd), in pole cells. We find that Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. We present evidence indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells.

The Drosophila Fragile X Protein dFMR1 Is Required During Early Embryogenesis for Pole Cell Formation and Rapid Nuclear Division Cycles

The FMR family of KH domain RNA-binding proteins is conserved from invertebrates to humans. In humans, inactivation of the X-linked FMR gene fragile X is the most common cause of mental retardation and leads to defects in neuronal architecture. While there are three FMR family members in humans, there is only a single gene, dfmr1, in flies. As in humans, inactivation of dfmr1 causes defects in neuronal architecture and in behavior. dfmr1 has other functions in the fly in addition to neurogenesis. Here we have analyzed its role during early embryonic development. We found that dfmr1 embryos display defects in the rapid nuclear division cycles that precede gastrulation in nuclear migration and in pole cell formation. While the aberrations in nuclear division are correlated with a defect in the assembly of centromeric/centric heterochromatin, the defects in pole cell formation are associated with alterations in the actin–myosin cytoskeleton.

Nanos downregulates transcription and modulates CTD phosphorylation in the soma of early Drosophila embryos.

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. We have previously reported that nos is required to keep the Sex-lethal establishment promoter, Sxl-Pe, off in the germline of both sexes. We show here that nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the soma as the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal domain (CTD) repeats of the large RNA polymerase II subunit. Finally, we present evidence indicating that the suppression of transcription in the soma by Nos protein is important for normal embryonic development.

Functional conservation of the sex-lethal sex determining promoter, Sxl-Pe, in Drosophila virilis.

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal (Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.