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The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

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Confocal analysis of eGFP expression in transgenic gonads. (A) The middle panel depicts the organisation of an Anopheles gambiae testis. In the apical tip the stem cell niche is sustained by a set of somatic cells, called hub cells (pink), which regulate the maintenance of the GSC (dark green) and SSC (dark blue) populations. Upon replication of the GSC, one of the two resulting daughter cells will differentiate into a primary spermatogonium (light green) and begin the developmental maturation process to become mature sperm. Testicular expression of eGFP from transgenic males of Vas1GFP (panels a, b), Vas2GFP (panels c, d) and β2-Tubulin-eGFP (panels e, f). Distribution of eGFP in the entire testes (panels a, c, e). Micrographs of the apical hub regions of transgenic testes (panels b, d, f). The eGFP expression in the GSC region is limited to Vas2GFP (panel d) and is absent in both Vas1GFP (panel b) and in β2-Tubulin-eGFP (panel f). (B) Expression of eGFP in ovarian follicles (panel g, h, i, j) and in germaria (panels k brightfield; l fluorescence). eGFP is expressed in nurse cells of all developing follicles and transported to the oocyte cytoplasm (panel g, i). In germaria eGFP clearly labels germline stem cells (green, GSCs) and developing cystocytes (light green) (panel l). Expression is not detectable in cells of the follicular epithelium (blue, FEC).
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Figure 3: Confocal analysis of eGFP expression in transgenic gonads. (A) The middle panel depicts the organisation of an Anopheles gambiae testis. In the apical tip the stem cell niche is sustained by a set of somatic cells, called hub cells (pink), which regulate the maintenance of the GSC (dark green) and SSC (dark blue) populations. Upon replication of the GSC, one of the two resulting daughter cells will differentiate into a primary spermatogonium (light green) and begin the developmental maturation process to become mature sperm. Testicular expression of eGFP from transgenic males of Vas1GFP (panels a, b), Vas2GFP (panels c, d) and β2-Tubulin-eGFP (panels e, f). Distribution of eGFP in the entire testes (panels a, c, e). Micrographs of the apical hub regions of transgenic testes (panels b, d, f). The eGFP expression in the GSC region is limited to Vas2GFP (panel d) and is absent in both Vas1GFP (panel b) and in β2-Tubulin-eGFP (panel f). (B) Expression of eGFP in ovarian follicles (panel g, h, i, j) and in germaria (panels k brightfield; l fluorescence). eGFP is expressed in nurse cells of all developing follicles and transported to the oocyte cytoplasm (panel g, i). In germaria eGFP clearly labels germline stem cells (green, GSCs) and developing cystocytes (light green) (panel l). Expression is not detectable in cells of the follicular epithelium (blue, FEC).

Mentions: Three independent transgenic A. gambiae lines were generated using the Vas1GFP construct. In all three transgenic lines the expression of eGFP was detectable exclusively in the vicinity of the developing gonads. Expression of eGFP was only detectable in approximately half of the transgenic larvae, irrespective of the sex of the transgenic parent. When separated and grown to adulthood all GFP positive larvae emerged as males and all GFP negative larvae as females (see Additional File 2A). To examine this phenotype further, RT-PCRs were performed on dissected adult tissues which revealed that eGFP transcript from Vas1GFP was only detectable in transgenic testes, unlike the endogenous vasa transcript which appeared in both testes and ovaries (Figure 2B). Exclusive activity of Vas1GFP in testes was further verified by western-blotting with anti-eGFP antibody (see Additional File 2D), confirming that absence of eGFP fluorescence in ovaries results from the absence of transcription from the Vas1GFP construct. To confirm this result we generated two Anopheles stephensi transgenic lines using the Vas1GFP construct (data not shown). Transgenics of this related vector species showed an identical eGFP expression pattern indicating that the Vas1GFP construct lacked the regulatory sequences required for transcription in female ovaries. The tissue specific expression pattern from Vas1GFP was similar to that reported for the A. gambiae testis specific promoter, β2-tubulin [21]. We compared eGFP fluorescence in developing larval stages and found that unlike the β2-tubulin promoter, in which testes-specific eGFP staining is only detectable in late 3rd instar larvae (L3), eGFP fluorescence from Vas1GFP was detectable in neonatal larvae (L1). Confocal analysis of dissected testes from Vas1GFP transgenic males revealed a widespread distribution of eGFP signal along the longitudinal axis of the organ (Figure 3A, panel a) ranging from the gonial amplification stages, developing spermatocysts up to individual mature sperm cells. Sperm transversing the vas efferens or removed from WT female spermathaeca showed cytoplasmic localization of eGFP (see Additional File 2B). When compared to testes from transgenic β2-tubulin eGFP reporter lines, the eGFP expression in Vas1GFP males was, albeit weaker overall, starting slightly closer to the hub of the organ (Figure 3A panels b, f), thus indicating that transcription (or translation) from Vas1GFP is likely initiated at an earlier stage of spermatogenesis than β2-tubulin. Vas1GFP expression was however not detectable in the apical tip of the testes indicating that the included regulatory regions did not direct expression in male germline stem cells (GSCs) (Figure 3A panel b).


The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Confocal analysis of eGFP expression in transgenic gonads. (A) The middle panel depicts the organisation of an Anopheles gambiae testis. In the apical tip the stem cell niche is sustained by a set of somatic cells, called hub cells (pink), which regulate the maintenance of the GSC (dark green) and SSC (dark blue) populations. Upon replication of the GSC, one of the two resulting daughter cells will differentiate into a primary spermatogonium (light green) and begin the developmental maturation process to become mature sperm. Testicular expression of eGFP from transgenic males of Vas1GFP (panels a, b), Vas2GFP (panels c, d) and β2-Tubulin-eGFP (panels e, f). Distribution of eGFP in the entire testes (panels a, c, e). Micrographs of the apical hub regions of transgenic testes (panels b, d, f). The eGFP expression in the GSC region is limited to Vas2GFP (panel d) and is absent in both Vas1GFP (panel b) and in β2-Tubulin-eGFP (panel f). (B) Expression of eGFP in ovarian follicles (panel g, h, i, j) and in germaria (panels k brightfield; l fluorescence). eGFP is expressed in nurse cells of all developing follicles and transported to the oocyte cytoplasm (panel g, i). In germaria eGFP clearly labels germline stem cells (green, GSCs) and developing cystocytes (light green) (panel l). Expression is not detectable in cells of the follicular epithelium (blue, FEC).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Confocal analysis of eGFP expression in transgenic gonads. (A) The middle panel depicts the organisation of an Anopheles gambiae testis. In the apical tip the stem cell niche is sustained by a set of somatic cells, called hub cells (pink), which regulate the maintenance of the GSC (dark green) and SSC (dark blue) populations. Upon replication of the GSC, one of the two resulting daughter cells will differentiate into a primary spermatogonium (light green) and begin the developmental maturation process to become mature sperm. Testicular expression of eGFP from transgenic males of Vas1GFP (panels a, b), Vas2GFP (panels c, d) and β2-Tubulin-eGFP (panels e, f). Distribution of eGFP in the entire testes (panels a, c, e). Micrographs of the apical hub regions of transgenic testes (panels b, d, f). The eGFP expression in the GSC region is limited to Vas2GFP (panel d) and is absent in both Vas1GFP (panel b) and in β2-Tubulin-eGFP (panel f). (B) Expression of eGFP in ovarian follicles (panel g, h, i, j) and in germaria (panels k brightfield; l fluorescence). eGFP is expressed in nurse cells of all developing follicles and transported to the oocyte cytoplasm (panel g, i). In germaria eGFP clearly labels germline stem cells (green, GSCs) and developing cystocytes (light green) (panel l). Expression is not detectable in cells of the follicular epithelium (blue, FEC).
Mentions: Three independent transgenic A. gambiae lines were generated using the Vas1GFP construct. In all three transgenic lines the expression of eGFP was detectable exclusively in the vicinity of the developing gonads. Expression of eGFP was only detectable in approximately half of the transgenic larvae, irrespective of the sex of the transgenic parent. When separated and grown to adulthood all GFP positive larvae emerged as males and all GFP negative larvae as females (see Additional File 2A). To examine this phenotype further, RT-PCRs were performed on dissected adult tissues which revealed that eGFP transcript from Vas1GFP was only detectable in transgenic testes, unlike the endogenous vasa transcript which appeared in both testes and ovaries (Figure 2B). Exclusive activity of Vas1GFP in testes was further verified by western-blotting with anti-eGFP antibody (see Additional File 2D), confirming that absence of eGFP fluorescence in ovaries results from the absence of transcription from the Vas1GFP construct. To confirm this result we generated two Anopheles stephensi transgenic lines using the Vas1GFP construct (data not shown). Transgenics of this related vector species showed an identical eGFP expression pattern indicating that the Vas1GFP construct lacked the regulatory sequences required for transcription in female ovaries. The tissue specific expression pattern from Vas1GFP was similar to that reported for the A. gambiae testis specific promoter, β2-tubulin [21]. We compared eGFP fluorescence in developing larval stages and found that unlike the β2-tubulin promoter, in which testes-specific eGFP staining is only detectable in late 3rd instar larvae (L3), eGFP fluorescence from Vas1GFP was detectable in neonatal larvae (L1). Confocal analysis of dissected testes from Vas1GFP transgenic males revealed a widespread distribution of eGFP signal along the longitudinal axis of the organ (Figure 3A, panel a) ranging from the gonial amplification stages, developing spermatocysts up to individual mature sperm cells. Sperm transversing the vas efferens or removed from WT female spermathaeca showed cytoplasmic localization of eGFP (see Additional File 2B). When compared to testes from transgenic β2-tubulin eGFP reporter lines, the eGFP expression in Vas1GFP males was, albeit weaker overall, starting slightly closer to the hub of the organ (Figure 3A panels b, f), thus indicating that transcription (or translation) from Vas1GFP is likely initiated at an earlier stage of spermatogenesis than β2-tubulin. Vas1GFP expression was however not detectable in the apical tip of the testes indicating that the included regulatory regions did not direct expression in male germline stem cells (GSCs) (Figure 3A panel b).

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

Show MeSH
Related in: MedlinePlus