Progress 04/01/13 to 03/31/18
Outputs Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?1) Publications in scientific journals are provided. 2) Dr. Tu has been giving presentations at international conferences and as invited seminar speakers 3) Dr. Tu has also given guest lectures to undergraduate and graduate students on this research. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts What was accomplished under these goals?
At the beginning of the 5 year project, we discovered four Y genes in An. stephensi (Criscione et al., 2013; Hall et al., 2013). Among these, Guy1 is the best candidate for the M factor because it encodes a small protein with predicted DNA-binding properties, its transcription starts at the very onset of embryonic development, and it does not require any other factor on the Y-chromosome for transcription initiation (Criscione et al., Insect molecular biology, 2013). During the last 4-5 years, we made significant progress towards understanding the function of Guy1 as a male determining factor, by demonstrating that the GUY1 protein confers 100% female-specific lethality and Guy1 transgenic males are reproductively more competitive than their non-transgenic siblings under laboratory conditions (Criscione et al., eLife, 2016) . During this reporting period, we further demonstrated the mechanism underlying the female-specific lethality, namely the mis-regulation of dosage compensation that results from the ectopic expression of Guy1 in XX females (Qi et al., eLife, under review). During this project period, we have also identified Y chromosome genes in the African malaria mosquito Anopheles gambiae and showed that the Y chromosomes underwent rapid remodeling within the past two million years (Hall et al., BMC genomics, 2014; Hall et al., PNAS, 2016). Using single molecule long read sequencing, we are improving the Y chromosome assembly of A. stephensi and A. gambiae, which will enable further evolutionary and functionary analysis of this important sex chromosome in mosquitoes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wu Y., Hu W., Bielder J., Chen X.G. and and Tu Z. 2018 Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi. Parasites & Vectors
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Progress 10/01/16 to 09/30/17
Outputs Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1) Students and other personnel served as mentors to train undergraduate students, graduate rotation students and under-represented students. 2) Trainedundergraduate students, graduate rotation students and under-represented students. 3) Students and other personnel presented their research at lab group, departmental and international meetings. How have the results been disseminated to communities of interest?1) Publicationin scientific journal. 2) Dr. Tu and students gave presentations at international conferences and at invited seminar speakers. 3) New genomic data, once carefully verified and analyzed, are being submitted to SRA for data sharing. 4) Dr. Tu has given guest lectures to undergraduate students and the general public. What do you plan to do during the next reporting period to accomplish the goals?We will follow the research design described in the proposal to achieve our objectives. Key activities in the next year will include: 1) Investigate the mechanism of Guy1 function using the newly obtained tagged-guy1 transgenic mosquitoes byperforming Chip-Seqto systematically identify Guy1 targets; 2) make new tagged-Guy2 transgenic lines to help identify Guy2 targets; 3)obtain new antibodies using the newly purified Guy1 and Guy2 recombinant proteins; 4) make additionaltet-off transgenic lines to overcome potential position effects of the transgene insertions.
Impacts What was accomplished under these goals?
During this reporting period, we have completed RNA-seq of transgenic lines to directly assess the involvement of Guy1 and another related gene, Guy2, on dosage compensation. We have performed analyses to show that both genes are involved in regulation of dosage compensation. We have now improved protein expression of both Guy1 and Guy2 and made new transgenic lines that have improved affinity tags to the Guy1 transgene. The new lines showed the same complete female lethal phenotype as previous lines. Therefore, we are in a better position to study the mechanism of Guy1 function. Another significant result in this reporting period is that we were able to expand our discovery and analysis of Y chromosomal genes to a small number of Anopheles species, which will allow us to study the evolution of Y genes and potentially apply what we learned to help control additional Anopheles vectors of malaria parasites.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Jiang X, Hall AB, Biedler JK, and Tu Z. 2017. Single Molecule RNA Sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi. Insect Mol Biol. DOI: 10.1111/imb.12294
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Progress 10/01/15 to 09/30/16
Outputs Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Students and other personnel published research articles. How have the results been disseminated to communities of interest?1) Publications in scientific journals and book chapters. 2) Dr. Tu has been giving presentations at international conferences and as invited seminar speakers. 3) Dr. Tu has also been engaging media outlets to disseminate information. 4) New genomic data, once carefully verified and analyzed, are submitted to SRA for data sharing. 5) Dr. Tu has also given guest lectures to undergraduate students and general public. What do you plan to do during the next reporting period to accomplish the goals?There is no change to our specific aims. We will follow the research design described in the proposal to achieve our objectives. Key activities in the next year will include: 1) continue RNA-seq of Guy1 transgenic lines; 2) obtain new Guy1 antibody; 3) perform Chip-Seq of Guy1 pull-down to systematically identify Guy1 targets; and 4) evaluate the new tet-off transgenic lines.
Impacts What was accomplished under these goals?
We have previously shown that a second Y-chromosome gene, sYG2 also conferred female-lethality. It is likely that Guy1 and sYG2 are components of a common signaling pathway. During this reporting period, we are performing RNA-seq of transgenic lines to directly assess the involvement of Guy1 and sYG2 on dosage compensation. These results indicate that sYG2 expression in females up-regulates X-linked genes, consistent with our hypothesis that sYG2 regulate dosage compensation by conferring hyper-expression of genes on the X chromosome. These results directly address the hypothesis of Aim 1 and it is the first direct evidence linking either Guy1 or sYG2 to dosage compensation and female lethality. We have also repeated the chromatin pull-down assay with additional controls using tagged Guy1 transgenic lines. We have now clearly shown that the Guy1 protein binds the sYG2 promoter specifically. This result is significant as it directly showed that Guy1 is a DNA-binding protein and it is thus very likely that Guy1 is a transcription factor that activates sYG2 and possibly other target genes. Another significant result in this reporting period is that we were able to purify the recombinant Guy1 protein in bacteria. We are in the process of making new antibodies using this recombinant protein. During this reporting period, we have also characterized Y chromosome genes and Y chromosome structure of Anopheles gambiae. These results provided the foundation for future comparative studies of Y genes in different Anopheles species.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Biedler JK, and Tu Z. 2016. Sex determination in mosquitoes. In (Editor: Raikhel, A.) Advances in Insect Physiology, Elsevier.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Criscione, F. Qi, Y., and Tu, Z. 2016. GUY1 confers complete female lethality and is a strong candidate for a male-determining factor in Anopheles stephensi. eLife, http://dx.doi.org/10.7554/eLife.19281.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Jiang X, Hall AB, Biedler JK, and Tu Z. 2016. Single Molecule RNA Sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi. Insect Mol Biol. Accepted.
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Progress 10/01/14 to 09/30/15
Outputs Target Audience:Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student was trained. How have the results been disseminated to communities of interest?The results were published in scientific journals and book chapters. Dr. Tu has been invited to discuss his results in scientific conferences and seminars. What do you plan to do during the next reporting period to accomplish the goals?1) Investigate the involvement of Guy1 in dosage compensation using embryonic RNAseq data 2) Verify the candidate target genes of Guy1 by reporter assays and promoter analysis 3) Using these composite transgenic lines, we now can knockout Guy1 and study the impact of such knockout.
Impacts What was accomplished under these goals?
In the past year, we have made significant advances that will enable us to complete the first two specific aims. First, after a number of optimizations, we were able to generate Illumina RNAseq libraries from single-embryo. We obtained single-embryo RNA-seq data and are in the process of obtaining more for statistical analyses. Such analyses will allow us to determine the impact of transgenic Guy1 expression at the molecular level (Aim 1). Second, we have generated a number of Guy1 transgenic lines including lines that express Guy1 from a composite gene (Guy1 open reading frame mixed with untranslated regions and promoters from other Anopheles stephensi genes). Thus, instead of knocking down Guy1 by RNAi as originally proposed (Aim 2), we can now use CRISPR/cas9 to knockout the native Guy1 without affecting the transgenic Guy1. This will mitigate potential sex-specific lethality associated with Guy1 knockout because male lethality will not occur until the transgene on the autosome and the mutated Y segregate.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Jiang X, Biedler JK, Qi Y, Hall AB, and Tu Z. 2015. Complete dosage compensation in Anopheles stephensi and the evolution of sex-biased genes in mosquitoes. Genome Biol Evol. 7, 1914-24.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2015
Citation:
Biedler, J.K.H., A.B.; Jiang, X.; Tu, Z. (2015) Exploring the sex-determination pathway for control of mosquito-borne infectious diseases. (Adelman, Z.N., ed), Elsevier, in press
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Progress 10/01/13 to 09/30/14
Outputs Target Audience: Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and the general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One postdoc was trained during the reporting period. How have the results been disseminated to communities of interest? The genome sequencing results were published. Dr. Tu was invited to scientific conferences and seminars to discuss the results. What do you plan to do during the next reporting period to accomplish the goals? 1) Continue working on the mechanism of the female lethality. Analysis the sequences of the transgenic and wild type embryos to identify genes that are affected by Guy1 expression. 2) Investiagte the target of Guy1 regulation by identifying the binding sites of the Guy1 protein in the early embryos. 3) Use CRISPR/cas9 to knockut Guy1 to investigate the impact of loss of Guy1.
Impacts What was accomplished under these goals?
We have recently uncovered four Y genes in An. stephensi (Criscione et al., 2013; Hall et al., 2013). Among these, Guy1 is the best candidate for the M factor because it encodes a small protein with predicted DNA-binding properties, its transcription starts at the very onset of embryonic development, and it does not require any other factor on the Y-chromosome for transcription initiation (Criscione et al., 2013). Multiple Guy1-expression transgenic lines were generated, which produced 6520 transgenic males and 0 transgenic females over multiple generations. A point mutation at the Guy1 initiation codon and a premature stop codon prior to the predicted DNA-binding domain abolished the male bias, indicating that the GUY1 protein is the cause of the male-only phenotype. Genotyping analysis showed that such a phenotype resulted from 100% lethality of XX individuals. Guy1 transgenic males were reproductively more competitive than their non-transgenic siblings under laboratory conditions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jiang, X., Peery, A., Hall, A.B., Sharakhov, I.V., and Tu, Z. 2014. Genome analysis of a major urban malaria mosquito Anopheles stephensi. Geome Biol., 15, 459.
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Progress 04/01/13 to 09/30/13
Outputs Target Audience: Scientists, students, educators, and the general public who are interested in insect sex-determination. Scientists and general public who are interested in developing genetic methods to control mosquito-borne infectious diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students participated in a mini-symposium on Vector-borne infectious diseases. Students and other personnel published research articles. How have the results been disseminated to communities of interest? Publications in peer-reviewed scientific journals: Hall, A.B., Qi , Y., Timoshevskiy, V. Sharakhova, M.V., Sharakhov, I.V. and Tu, Z. 2013. Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females. BMC Genomics, 14, 273 (13 print pages). Criscione, Z. Qi, Y., Saunders, T.R., Hall, B. and Tu, Z. 2013. A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development. Insect Mol Biol., 22, 433-441. What do you plan to do during the next reporting period to accomplish the goals? 1. We hypothesize that Guy1 is involved in both sex-determination and dosage compensation, similar to the Drosophila sex-lethal gene. To further determine the molecular mechanism of Guy1 function, we will perform RNA-seq or targeted analysis of doublesex transcripts on genotyped embryos (Crisione et al., 2013). We will also compare transcription of genes in the X with genes in the autosome. We have also demonstrated the feasibility of using Tet-off and Gal4/UAS to confer conditional gene expression in A. stephensi embryos. We will develop additional Guy1 expression lines based on gal4/uas conditional control. 2. Because the recent success of TALEN-based gene knockout in mosquitoes, which was described as an alternative in our proposal, we have now adopted the TALEN approach to achieve Guy1 knockout instead of the shRNA-based Guy1 knockdown. We have made three TALEN constructs that specifically target Guy1 and will perform transgenic experiments. We are also exploring the CRISPR-based method to knockout Guy1.
Impacts What was accomplished under these goals?
During this reporting period, we further characterized Guy1 and three additional Y genes in Anopheles stephensi and the results have now been published (Crisione et al., 2013; Hall et al., 2013). We then showed that embryonic expression of the Guy1 gene introduced dramatic male bias and obtained Guy1-expressing transgenic A. stephensi strains that produced all male or nearly all male transgenics. This is potentially exciting because these results indicate that Guy1 is possibly the first male-determining factor found on an insect Y chromosome. We are investigating the molecular mechanism of Guy1 function. We have also demonstrated the feasibility of using Tet-off and Gal4/UAS to confer conditional gene expression in A. stephensi embryos, which will facilitate the production of stable homozygous Guy1 transgenic lines that may improve sex separation and mosquito control methods. We are in the process of producing Guy1 knockout lines using site-specific nucleases.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hall, A.B., Qi , Y., Timoshevskiy, V. Sharakhova, M.V., Sharakhov, I.V. and Tu, Z. 2013. Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females. BMC Genomics, 14, 273 (13 print pages).
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Criscione, Z. Qi, Y., Saunders, T.R., Hall, B. and Tu, Z. 2013. A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development. Insect Mol Biol., 22, 433-441.
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