Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus

Abstract

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions we made transgenic flies expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1–6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation.

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal D

“…A previously described BicD::GFP construct rescued the sterility phenotype and the embryonic developmental arrest of BicD loss-of-function mutant females (BicD::GFP, BicD null homozygous females) (Paré and Suter, 2000) (Fig. 2D-E).…”

“…Thus, we designed a strategy to knock down the BicD protein directly in young embryos (stage 1) using the deGradFP technique (degrade Green Fluorescent Protein), a method to target fusion proteins with GFP for destruction or inactivation (Caussinus et al, 2011). For this, we took advantage of the functional, genomic BicD::GFP construct (Paré and Suter, 2000). Additionally, we constructed a deGradFP system specifically active during embryogenesis but not oogenesis by expressing the deGradFP (NSlmb-vhhGFP4) from a hunchback (hb) minimal maternal promoter coupled with the bcd 3'-UTR (Fig.…”

“…(Exelixis) was used as BicD deficiency (BicD Df ). The BicD null allele, BicD r5 , Bic-D mom , and BicD::GFP were described (Paré and Suter, 2000;Ran et al, 1994;Swan and Suter, 1996;Vazquez-Pianzola et al, 2014). White (w) flies were used as controls.…”

The Importance of the Position of the Nucleus in Drosophila Oocyte Development

“…Nuclear positioning occurs between stages 5 and 7, with the nucleus assuming an asymmetric position at later stages [18,22,23,41]. A peculiarity of the oocyte nuclear migration is that it is a three-dimensional process.…”