The microtubule array that is generated acts as a track for dynein motors to carry cargos towards the anterior end of the oocyte, i.e. towards the nurse cells. The nucleus of the oocyte travels along the microtubule array via a dynein motor. Depending on the side of the microtubule track that the nucleus travels along, whichever side it ends up on will be the dorsal side. The dorsalized cell fate is determined by localization of Gurken, which is expressed by gurken mRNA at the oocyte nucleus. When the nucleus moves to a particular side, gurken protein activates torpedo, which causes follicle cells to differentiate into a dorsal morphology, by blocking the default ventral fate of follicle cells.
CSN5/Jab1 mutations affect axis formation in the Drosophila oocyte by activating a meiotic checkpoint (3)Edit
One paper done at University of California, Berkeley looked at the effects of CSN5/Jab1 mutations on the accumulation of the Gurken protein in the drosophila oocyte, showing that Gurken protein accumulation is in fact required for axis determination in the developing oocyte. This paper showed that CSN5 mutations block gurken acummulation in the oocyte and also that these mutations modify Vasa, known to be involved in spindle formation and meiotic repair mechanisms, which is required for gurken translation. The resulting CSN5 mutant phenotype that was observed by this particular group was similar to that of spindle-class genes required to repair double-strand breaks occurring during meiotic recombination. When the breaks are left unrepaired, a check-point controlled by mei-41 is activated. It was shown that the CSN5 phenotype (reduced Gurken accumulation, defective axis formation, and modified Vasa) was suppressed by mei-41 mutations or by mutations in mei-W68, which is required for double strand break formation.
They used flies carrying a GFP balancer to determine the lethal stage of development. Eggs were collected and were examined and females were dissected to confirm the presence of homozygous-mutant follicle cells characterized by the absence of GFP. They visualized egg chambers and embyros by immunostaining with antibodies against Gurken, Sperm-Tail and Vasa. To monitor the lacZ expression of the P-lacZ insertion mutations, ovaries were stained with rhodamine-conjugated phalloidin. DAPI was used to visualize the nuclei. In situ hybridization was used with digoxigenin-labeled antisense RNA probes. Signals were visualized by histochemical staining with alkaline phosphatase.
Western blots were also used. CSN5/JAB1 protein was detected using a mouse polyclonal and 3 mouse monoclonal antibodies against mouse Jab1 because all of these antibodies recognized the same 37-38kDa band, which is the predicted size of Drosophila CSN5.
ResultsEditFirst, they showed that CSN5 is expressed in nurse cells during oogenesis. This was confirmed via in situ hybridization, showing the accumulation of CSN5 RNA within the nurse cells during most of oogenesis, until stage 10 when CSN5 mRNA is transferred to the oocyte when most of the nurse cell cytoplasm are also transferred (Figure 3). In A and B, it is clear that CSN5 expression in wild type ovaries is present in nurse cells. C and D are reflective of mutant germline clones, and depict significantly decreased expression of CSN5. E shows an early wild type embryo with complete expression of CSN5, suggesting that it has a strong maternal contribution. F shows that CSN5 heterozygotes lack maternal CSN5 mRNA but that zygotic expression is present (arrow). G and H illustrate that CSN5 RNA is most strongly expressed in the ventral furrow, the cephalic furrow, and the anterior and posterior midgut invaginations. The Western blot in I shows wild type versus CSN5 mutant ovarian extracts.
Next, it was shown that CSN5 is required for eggshell patterning. To enable analysis of early embryonic requirements for CSN5, they induced homozygous CSN5 mutant germline clones which revealed requirements for CSN5 during oogenesis as well as embryogenesis. In ovarian germline clones, the level of CSN5 RNA is significantly reduced, but still detectable, indicating that the P-element induced allele is hypomorphic (Figure 1C, D). Depending on the paternal allele, embryos from the germline clones showed either a reduced amount of CSN5 RNA in the zygotic pattern (fig.1F) or no detectable CSN5 RNA. Those carrying the CSN5 germline clones laid eggs with a range of abnormal phenotypes that were affected by temperature (Table 1 ). Flies laid at 25 deg. C were the closest to normal. The most frequent defects at 18 deg. C were different than those at 29 deg. C, which can be explained by the fact that abberrations in patterning the follicular epithelium predominate at 18 deg while defects in follicle cell migration predominate at 29 deg. Figure 2 compares the defects to 2A which depicts a wild type embryo with normal dorsal appendages (DA). Figure 2B-F show eggshells dervied from CSN5-mutant females consisting of fused, absent, or duplicate DA and abnormally shaped and weak eggshells.
Because the eggshell phenotypes were only partially penetrant, they could have been caused by somatic cells, and not germline CSN5 clones. To refute this, they induced somatic clones in the ovary by using the follicle cell driver E22c-GAL4 to induce expression of UAS-FLP and say no eggshell defects at any temperature, indicating that CSN5 function is required only in the germline and thus essential for proper development.
It was then noticed that some mutations interrupting the Dorsal/Ventral (DV) patterning of the eggshell also affect the patterning of the embryo. To look for effects on the DV fate map, they used markers for 3 zygotic genes: dpp, rho, twi (Figure 3 ). For all three markers, many of the CSN5 mutants appeared to be ventralized. In these embryos, dpp expression on the dorsal side was reduced or absent. The dorsal rho stripe was also reduced and the lateral stripes were removed on the dorsal side. Twi expression expanded dorsally. Finally, some embryos showed stronger ventralization at their anterior or posterior.
Other results that were obtained include:
- CSN5 required for anterior-posterior polarization: They examined the spatial localization of bicoid (bcd) and oskar (osk) RNA, which represent the anterior and posterior sides of the embryo, respectively. As previously discussed, bicoid RNA localization at the anterior side of the oocyte is important for developing the microtubule array which subsequently leads to dorsal/ventral specification and also for anterior/posterior determination. In the abnormal CSN5 mutant oocytes, the bicoid mRNA was distributed diffusely throughout (and sometimes centrally) the oocyte (Figure 4B ). In mutant embryos, the bicoid mRNA often shifted towards the dorsal side of the embryo.
- CSN5 may also be required for proper pole cell organization
- CSN5 is required for Gurken (grk) signalling (Figure 5 ): They used reporters for either the posterior or dorsal Grk signal to assess the role of CSN5 in Grk signaling. In the absence of posterior Grk signal, the posterior follicle cells adopt the anterior follicle cell fate and express markers characteristic of border cells. They used two of these markers (Figure 5A and 5C) to monitor whether CSN5 is required for the early Grk signal. For both markers, the loss of CSN5 from the germline caused lacZ expression in the posterior follicle cells, suggesting a reduction in Grk signaling (fig. 5B and D). Figure 6 shows that CSN5 is necessary for Grk protein expression, not for grk transcription.
1 & 2: Developmental Biology 9th edition by Scott F. Gilbert. 2010. Sinauer Associates, Inc. Sunderland, MA
3. UC Berkeley Paper: dev.biologists.org/content/129/21/5053.long