RNA localization in the honeybee (Apis mellifera) ooctye reveals insights about the evolution of RNA localization mechanism

This page is based on Wilson, M. J., & Dearden, P. K. (2013) paper, RNA localization in the honeybee (Apis mellifera) ooctye reveals insights about the evolution of RNA localization mechanism

Honeybees are increasingly being used as a model organism to understand development because controlling the exact genetic makeup of colonies is feasible with modern techniques and queen bees can lay up to 1500 eggs a day!

For more overview information about honeybees, go to this link, or watch this brief video about bee development.

What is being researched?

In order to ascertain axes, early embryos of many animals asymmetrically distribute transcripts encoding patterning factors. mRNAs in many insects are provided maternally and so are genes that are important in axis specification in early embryo development which are localized in areas of the oocyte. In Drosophila, bicoid has been identified as a gene that determines the anterior region and gurken, which signals anterior-dorsal formation (this video briefly explains early development of Drosophila including axis patterning). However in honeybees, orthologues of these genes that are so important in axis patterning are missing, including oskar which is essential in establishing germline in other insects.

Honeybees use maternal RNA localization extensively based on previous research that showed oocytes and early embryos have localized transcripts of several patterning genes. The researchers examined the localization of RNA in honeybees by seeing if mechanisms that recognize transcripts in Drosophila will do so in honeybees.

Physical comparison between the fruit fly and honeybee

Major Findings

Identification of potential patterning gene transcripts that are localized in honeybee queen oocytes

Researchers used honeybee queen oocytes as they are large and abundant in order to understand which genes’ RNAs are localized to anterior and posterior regions. By using microarrays, they were able to identify transcripts in honeybees that are differentially localized and select a later stage oocyte since the asymmetry of localized RNAs in early embryos is the target of this research. 24 genes were cloned and labeled with probes in situ. They found two transcripts that showed enrichment in both poles of the oocyte and transcripts that were enriched at either pole, also exhibited asymmetric distribution in mature oocytes (fig 1).

Fig 1. Validation of RNA localization by in situ hybridization. Labeled RNA probes were used to determine the expression of transcripts identified in microarray analysis as being enriched to the poles of the oocyte. Transcripts from the microarray analysis that were identified as being anteriorly enriched were found to be confined to the anterior half of the embryo. Stage 7–8 oocytes are shown with anterior at the left and dorsal side up.

Lack of conservation of RNA distribution between orthologous transcripts

The researchers wanted to see if these transcripts were evolutionarily conserved so they looked at twelve orthologue RNA expression of Drosophila that are supposed to be localized in honeybees. The results showed that only 3/16 of the transcripts in Drosophila were distributed asymmetrically in oocytes (fig 3) thus showing little conservation between Drosophila and Aphis orthologous transcripts.

Fig 3. Ovary expression of Drosophila orthologues of localized Apis transcripts. Detection of Drosophila orthologues of localized Apis transcripts in the Drosophila egg chamber. (A) Expression of Drosophila transcripts in late stage egg chambers. These transcripts were expressed and RNA detected in the nurse cells and oocyte, but no localization of these transcripts was found. (B) Three Drosophila RNA transcripts that were found to be localized in Drosophila egg chambers.

Evolutionarily conserved recognition of localization sequences

A total of four transcripts that are localized in honeybees were injected into honeybee and Drosophila embryos; two were orthologues that are also localized in Drosophila (tsu and PkaC1) and the other two were expressed but not localized in Drosophila (GB13806 and GB17282). After being injecting the oocytes with tsu, it distributed from anterior to posterior in stage 5 but was restricted in the anterior at stage 8. GB13806 distributed throughout and was enriched at the anterior region. In the beginning, GB17282 was enriched on one side but later became enriched on the posterior pole by stage 8. PkaC1 localized in the posterior pole throughout stage 5 and stage 8. This shows that RNAs contain localization signals that determine which axis these transcripts are to locate to (fig 4).

Fig. 4. Localization of fluorescently labeled capped-RNAs in honeybee oocytes. In vitro synthesized transcribed labeled RNAs were injected into the nurse cells of stage 2–4 oocytes and examined following incubated for 5 h at 35 1C. Honeybee oocytes are shown anterior to the left and dorsal side up. Stage 9 Drosophila egg chambers are shown anterior to the left and dorsal side up. Abbreviations: oocyte nucleus (ON).

Conclusion

The purpose of this study was to investigate the localization of RNA in honeybee oocytes by examining the pattern of evolution and this was achieved well by conducting the above experiments with each individual experiment building on to the next thus connecting the study together and reaching to a conclusion.

They were able to conclude that localizing RNA via the machinery and signal recognition processes are conserved but RNA complements that are transported and localized undergo rapid evolution. The implications of these findings can be applied to the evolution of axis formation for insects; in fruit flies, bicoid determines anterior patterning whereas bicoid-lacking honeybees use a different set of localized transcripts. Therefore a larger group of transcripts supplies RNAs that break the oocyte symmetry and based from this new found information, axis formation may come from new patterning molecules and with further research, can be discovered.

References

Wilson, M. J., & Dearden, P. K. (2013). RNA localization in the honeybee (Apis mellifera) ooctye reveals insights about the evolution of RNA localization mechanism. Developmental Biology, 375(2), 193-201.

http://www.youtube.com/watch?v=BTQQ8lPl6G8

http://www.cyberbee.net/research.htm

http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Student/fly_webo4/web%20presentation/fly1

http://www.nzdl.org/gsdlmod?e=d-00000-00—off-0fnl2.2–00-0—-0-10-0—0—0direct-10—4——-0-1l–11-en-50—20-about—00-0-1-00-0–4—-0-0-11-10-0utfZz-8-00&cl=CL3.50&d=HASHdf971f1d6cfb2110b7ce5d.6&gt=1

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