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A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

Fagegaltier D, König A, Gordon A, Lai EC, Gingeras TR, Hannon GJ, Shcherbata HR - Genetics (2014)

Bottom Line: Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation.Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads.This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 fagegalt@cshl.edu halyna.shcherbata@mpibpc.mpg.de.

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let-7 deficiency affects somatic cells behavior in ovaries and testes, which cell-non-autonomously influences early germline differentiation. Drosophila ovaries and testes present commonalities in their general organization and the type of cells they comprise (Fuller and Spradling 2007). The ovary is a paired organ consisting of 16–20 ovarioles, each representing a string of progressively developing egg chambers. (A) At the apex of an ovariole, the germarium comprises somatic cells (terminal filament, TF; cap cells, CpC’s; escort cells, ECs; follicle stem cells, FSCs; follicle cells, FCs) and germline cells (germline stem cells, GSCs; cystoblast, CB; differentiating cyst, Cyst). GSCs are physically attached to the somatic cluster of cap cells that, with the terminal filament cells, represent the GSC niche. GSCs divide into a differentiating cystoblast, which then divides four times with incomplete cytokinesis, producing a 16-cell cyst in which 1 cell becomes the oocyte while the others develop as nurse cells. These different germline cell populations can be easily identified by their location and specific markers in the germarium. For example, Drosophila adducin homolog antibody marks an actin-rich cellular organelle represented as a dot-like structure in single cells (GSCs and CBs) and as a branched fusome in the developing cysts. There is another class of somatic cells at the anterior of the germarium, called the escort cells. These squamous cells are mitotically quiescent and envelop differentiating cysts to protect them from niche signaling, an important role for germline differentiation (Chen et al. 2011). ECs guide differentiating cysts to the posterior end of the germarium, where the germline becomes encapsulated by the follicular epithelium and pinched off from the germarium. (B) The Drosophila testes are a paired tubular organ that consists of somatic and germline cells. Scheme depicting the testis apex somatic cells (hub cells, Hub; cyst stem cells, CySC’s; cyst stem cells lineage, CySC lineage) and germline cells (germline stem cells, GSCs; gonioblast, GB; differentiating spermatocysts and spermatogonia). Attached to the stem cell niche, termed the hub, reside two types of stem cells: GSCs and CySC’s. While the hub is made by a cluster of postmitotic somatic cells, both stem cell types divide in synchrony to produce differentiating germline cysts, each of which is encapsulated by two somatic CySC’s lineage cells. This encapsulation is critical for proper germline differentiation (Leatherman and Dinardo 2010). Similar to the ovarian GSC progeny, the germline gonioblast undergoes four rounds of incomplete cytokinesis to produce 16 primary spermatocytes in a cyst, eventually generating 64 sperm cells. (C) The relative expression levels of miRNA let-7 in female and male carcasses, ovaries, and testes show that let-7 miRNA is sex biased and expressed at the higher levels in testes (see also Table S17). (D, D′, E, E′) Localization of let-7-C miRNAs in the ovaries and testes, detected via membrane GFP and nuclear lacZ expressed under the control of the let-7-C promoter (let-7-CGK1–Gal4/UAS–CD8-GFP::nuc–lacZ). In the ovary, let-7-C is expressed in the somatic cells of the germarium, CpCs and ECs (D). (D′) More ECs express let-7-C, shown by the presence of lacZ (β-Gal, green) when adult flies were subject to a heat shock for 1 hr prior to dissection. (E and E′) In the testis, let-7-C is broadly expressed in all somatic cells, CySC’s and their lineage and the hub cells (let-7-CGK1-Gal4/UAS-CD8-GFP::nuc-lacZ). (F and F′) Control let-7-C rescue germaria (P{W8, let-7-C}/+; let-7-CGK1/KO1) show typical numbers of germline SSCs and developing cysts (marked by the spectrosome and fusome marker Adducin, Add, red) as well as somatic ECs (marked by Traffic jam, Tj, green). The GSC niche (marked by Lamin C, LamC, red) is outlined by yellow dashed lines. (G and G′) Somatic cell ∆let-7 clones (Ubi–GFP, FRT40A/ FRT40A, let-7 miR-125; bab1–Gal4, UAS–Flp/P{W8, let-7-CΔlet-7}) are marked by the absence of GFP and outlined by white dashed lines. Upon let-7 depletion in the soma of the germarium, the number of somatic ECs and germline SSCs increases, while the number of fusome-containing cysts decreases. The magnification is the same in F and G. (H) The testicular apex of a control let-7-C rescue (P{W8, let-7-C}; let-7-CGK1/KO1) displays typical numbers of germline CBs and cysts (marked by spectrosome and fusome marker Add, red) and the CySC lineage cells (marked by Tj, green); yellow dashed lines outline the hub (marked by Fasciclin III, Fas3, red). (J) In Δlet-7 mutant testis (let-7-CGK1/KO1; P{W8, let-7-CΔlet-7}/+) the CySC lineage cells cluster in larger groups and express the ovarian follicular epithelium marker, Fas3. These clusters (outlined by white dashed lines) can be found at the apex or the side of the testicular tube. (I) Percentages of mutant testes containing large ≥5 or >10 somatic cell clusters in comparison to control let-7-C Rescue. Ten to 20 testes were analyzed for each genotype. Red: LamC + Add in D, D′, F, F′, G, G′ ; Fas3 + Add in E, H, J, J′. Green: GFP + β-Gal in D and E; GFP in G, G′; Tj in F, F′, H, J, J′. White: GFP + β-Gal in E′. Blue: DAPI in D–H, G–J′. D, E + E′, H, J + J′ are single confocal sections while D′, F + F′, and G + G′ are projections of several z-stacks.
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fig3: let-7 deficiency affects somatic cells behavior in ovaries and testes, which cell-non-autonomously influences early germline differentiation. Drosophila ovaries and testes present commonalities in their general organization and the type of cells they comprise (Fuller and Spradling 2007). The ovary is a paired organ consisting of 16–20 ovarioles, each representing a string of progressively developing egg chambers. (A) At the apex of an ovariole, the germarium comprises somatic cells (terminal filament, TF; cap cells, CpC’s; escort cells, ECs; follicle stem cells, FSCs; follicle cells, FCs) and germline cells (germline stem cells, GSCs; cystoblast, CB; differentiating cyst, Cyst). GSCs are physically attached to the somatic cluster of cap cells that, with the terminal filament cells, represent the GSC niche. GSCs divide into a differentiating cystoblast, which then divides four times with incomplete cytokinesis, producing a 16-cell cyst in which 1 cell becomes the oocyte while the others develop as nurse cells. These different germline cell populations can be easily identified by their location and specific markers in the germarium. For example, Drosophila adducin homolog antibody marks an actin-rich cellular organelle represented as a dot-like structure in single cells (GSCs and CBs) and as a branched fusome in the developing cysts. There is another class of somatic cells at the anterior of the germarium, called the escort cells. These squamous cells are mitotically quiescent and envelop differentiating cysts to protect them from niche signaling, an important role for germline differentiation (Chen et al. 2011). ECs guide differentiating cysts to the posterior end of the germarium, where the germline becomes encapsulated by the follicular epithelium and pinched off from the germarium. (B) The Drosophila testes are a paired tubular organ that consists of somatic and germline cells. Scheme depicting the testis apex somatic cells (hub cells, Hub; cyst stem cells, CySC’s; cyst stem cells lineage, CySC lineage) and germline cells (germline stem cells, GSCs; gonioblast, GB; differentiating spermatocysts and spermatogonia). Attached to the stem cell niche, termed the hub, reside two types of stem cells: GSCs and CySC’s. While the hub is made by a cluster of postmitotic somatic cells, both stem cell types divide in synchrony to produce differentiating germline cysts, each of which is encapsulated by two somatic CySC’s lineage cells. This encapsulation is critical for proper germline differentiation (Leatherman and Dinardo 2010). Similar to the ovarian GSC progeny, the germline gonioblast undergoes four rounds of incomplete cytokinesis to produce 16 primary spermatocytes in a cyst, eventually generating 64 sperm cells. (C) The relative expression levels of miRNA let-7 in female and male carcasses, ovaries, and testes show that let-7 miRNA is sex biased and expressed at the higher levels in testes (see also Table S17). (D, D′, E, E′) Localization of let-7-C miRNAs in the ovaries and testes, detected via membrane GFP and nuclear lacZ expressed under the control of the let-7-C promoter (let-7-CGK1–Gal4/UAS–CD8-GFP::nuc–lacZ). In the ovary, let-7-C is expressed in the somatic cells of the germarium, CpCs and ECs (D). (D′) More ECs express let-7-C, shown by the presence of lacZ (β-Gal, green) when adult flies were subject to a heat shock for 1 hr prior to dissection. (E and E′) In the testis, let-7-C is broadly expressed in all somatic cells, CySC’s and their lineage and the hub cells (let-7-CGK1-Gal4/UAS-CD8-GFP::nuc-lacZ). (F and F′) Control let-7-C rescue germaria (P{W8, let-7-C}/+; let-7-CGK1/KO1) show typical numbers of germline SSCs and developing cysts (marked by the spectrosome and fusome marker Adducin, Add, red) as well as somatic ECs (marked by Traffic jam, Tj, green). The GSC niche (marked by Lamin C, LamC, red) is outlined by yellow dashed lines. (G and G′) Somatic cell ∆let-7 clones (Ubi–GFP, FRT40A/ FRT40A, let-7 miR-125; bab1–Gal4, UAS–Flp/P{W8, let-7-CΔlet-7}) are marked by the absence of GFP and outlined by white dashed lines. Upon let-7 depletion in the soma of the germarium, the number of somatic ECs and germline SSCs increases, while the number of fusome-containing cysts decreases. The magnification is the same in F and G. (H) The testicular apex of a control let-7-C rescue (P{W8, let-7-C}; let-7-CGK1/KO1) displays typical numbers of germline CBs and cysts (marked by spectrosome and fusome marker Add, red) and the CySC lineage cells (marked by Tj, green); yellow dashed lines outline the hub (marked by Fasciclin III, Fas3, red). (J) In Δlet-7 mutant testis (let-7-CGK1/KO1; P{W8, let-7-CΔlet-7}/+) the CySC lineage cells cluster in larger groups and express the ovarian follicular epithelium marker, Fas3. These clusters (outlined by white dashed lines) can be found at the apex or the side of the testicular tube. (I) Percentages of mutant testes containing large ≥5 or >10 somatic cell clusters in comparison to control let-7-C Rescue. Ten to 20 testes were analyzed for each genotype. Red: LamC + Add in D, D′, F, F′, G, G′ ; Fas3 + Add in E, H, J, J′. Green: GFP + β-Gal in D and E; GFP in G, G′; Tj in F, F′, H, J, J′. White: GFP + β-Gal in E′. Blue: DAPI in D–H, G–J′. D, E + E′, H, J + J′ are single confocal sections while D′, F + F′, and G + G′ are projections of several z-stacks.

Mentions: We analyzed let-7-C expression pattern in the adult Drosophila gonads using Gal4 under the control of the intrinsic let-7-C promoter. In the germarium, let-7-C is expressed in some of the somatic escort (ECs) and cap cells (CpCs; Figure 3, A, B, and D). The number of let-7-C-expressing cells and the activity of the let-7-C promoter per se fluctuate in CpCs and ECs in different germaria and within the same germarium (compare Figure 3, D and D′), demonstrating that the expression pattern of let-7-C is highly dynamic.


A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

Fagegaltier D, König A, Gordon A, Lai EC, Gingeras TR, Hannon GJ, Shcherbata HR - Genetics (2014)

let-7 deficiency affects somatic cells behavior in ovaries and testes, which cell-non-autonomously influences early germline differentiation. Drosophila ovaries and testes present commonalities in their general organization and the type of cells they comprise (Fuller and Spradling 2007). The ovary is a paired organ consisting of 16–20 ovarioles, each representing a string of progressively developing egg chambers. (A) At the apex of an ovariole, the germarium comprises somatic cells (terminal filament, TF; cap cells, CpC’s; escort cells, ECs; follicle stem cells, FSCs; follicle cells, FCs) and germline cells (germline stem cells, GSCs; cystoblast, CB; differentiating cyst, Cyst). GSCs are physically attached to the somatic cluster of cap cells that, with the terminal filament cells, represent the GSC niche. GSCs divide into a differentiating cystoblast, which then divides four times with incomplete cytokinesis, producing a 16-cell cyst in which 1 cell becomes the oocyte while the others develop as nurse cells. These different germline cell populations can be easily identified by their location and specific markers in the germarium. For example, Drosophila adducin homolog antibody marks an actin-rich cellular organelle represented as a dot-like structure in single cells (GSCs and CBs) and as a branched fusome in the developing cysts. There is another class of somatic cells at the anterior of the germarium, called the escort cells. These squamous cells are mitotically quiescent and envelop differentiating cysts to protect them from niche signaling, an important role for germline differentiation (Chen et al. 2011). ECs guide differentiating cysts to the posterior end of the germarium, where the germline becomes encapsulated by the follicular epithelium and pinched off from the germarium. (B) The Drosophila testes are a paired tubular organ that consists of somatic and germline cells. Scheme depicting the testis apex somatic cells (hub cells, Hub; cyst stem cells, CySC’s; cyst stem cells lineage, CySC lineage) and germline cells (germline stem cells, GSCs; gonioblast, GB; differentiating spermatocysts and spermatogonia). Attached to the stem cell niche, termed the hub, reside two types of stem cells: GSCs and CySC’s. While the hub is made by a cluster of postmitotic somatic cells, both stem cell types divide in synchrony to produce differentiating germline cysts, each of which is encapsulated by two somatic CySC’s lineage cells. This encapsulation is critical for proper germline differentiation (Leatherman and Dinardo 2010). Similar to the ovarian GSC progeny, the germline gonioblast undergoes four rounds of incomplete cytokinesis to produce 16 primary spermatocytes in a cyst, eventually generating 64 sperm cells. (C) The relative expression levels of miRNA let-7 in female and male carcasses, ovaries, and testes show that let-7 miRNA is sex biased and expressed at the higher levels in testes (see also Table S17). (D, D′, E, E′) Localization of let-7-C miRNAs in the ovaries and testes, detected via membrane GFP and nuclear lacZ expressed under the control of the let-7-C promoter (let-7-CGK1–Gal4/UAS–CD8-GFP::nuc–lacZ). In the ovary, let-7-C is expressed in the somatic cells of the germarium, CpCs and ECs (D). (D′) More ECs express let-7-C, shown by the presence of lacZ (β-Gal, green) when adult flies were subject to a heat shock for 1 hr prior to dissection. (E and E′) In the testis, let-7-C is broadly expressed in all somatic cells, CySC’s and their lineage and the hub cells (let-7-CGK1-Gal4/UAS-CD8-GFP::nuc-lacZ). (F and F′) Control let-7-C rescue germaria (P{W8, let-7-C}/+; let-7-CGK1/KO1) show typical numbers of germline SSCs and developing cysts (marked by the spectrosome and fusome marker Adducin, Add, red) as well as somatic ECs (marked by Traffic jam, Tj, green). The GSC niche (marked by Lamin C, LamC, red) is outlined by yellow dashed lines. (G and G′) Somatic cell ∆let-7 clones (Ubi–GFP, FRT40A/ FRT40A, let-7 miR-125; bab1–Gal4, UAS–Flp/P{W8, let-7-CΔlet-7}) are marked by the absence of GFP and outlined by white dashed lines. Upon let-7 depletion in the soma of the germarium, the number of somatic ECs and germline SSCs increases, while the number of fusome-containing cysts decreases. The magnification is the same in F and G. (H) The testicular apex of a control let-7-C rescue (P{W8, let-7-C}; let-7-CGK1/KO1) displays typical numbers of germline CBs and cysts (marked by spectrosome and fusome marker Add, red) and the CySC lineage cells (marked by Tj, green); yellow dashed lines outline the hub (marked by Fasciclin III, Fas3, red). (J) In Δlet-7 mutant testis (let-7-CGK1/KO1; P{W8, let-7-CΔlet-7}/+) the CySC lineage cells cluster in larger groups and express the ovarian follicular epithelium marker, Fas3. These clusters (outlined by white dashed lines) can be found at the apex or the side of the testicular tube. (I) Percentages of mutant testes containing large ≥5 or >10 somatic cell clusters in comparison to control let-7-C Rescue. Ten to 20 testes were analyzed for each genotype. Red: LamC + Add in D, D′, F, F′, G, G′ ; Fas3 + Add in E, H, J, J′. Green: GFP + β-Gal in D and E; GFP in G, G′; Tj in F, F′, H, J, J′. White: GFP + β-Gal in E′. Blue: DAPI in D–H, G–J′. D, E + E′, H, J + J′ are single confocal sections while D′, F + F′, and G + G′ are projections of several z-stacks.
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Related In: Results  -  Collection

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fig3: let-7 deficiency affects somatic cells behavior in ovaries and testes, which cell-non-autonomously influences early germline differentiation. Drosophila ovaries and testes present commonalities in their general organization and the type of cells they comprise (Fuller and Spradling 2007). The ovary is a paired organ consisting of 16–20 ovarioles, each representing a string of progressively developing egg chambers. (A) At the apex of an ovariole, the germarium comprises somatic cells (terminal filament, TF; cap cells, CpC’s; escort cells, ECs; follicle stem cells, FSCs; follicle cells, FCs) and germline cells (germline stem cells, GSCs; cystoblast, CB; differentiating cyst, Cyst). GSCs are physically attached to the somatic cluster of cap cells that, with the terminal filament cells, represent the GSC niche. GSCs divide into a differentiating cystoblast, which then divides four times with incomplete cytokinesis, producing a 16-cell cyst in which 1 cell becomes the oocyte while the others develop as nurse cells. These different germline cell populations can be easily identified by their location and specific markers in the germarium. For example, Drosophila adducin homolog antibody marks an actin-rich cellular organelle represented as a dot-like structure in single cells (GSCs and CBs) and as a branched fusome in the developing cysts. There is another class of somatic cells at the anterior of the germarium, called the escort cells. These squamous cells are mitotically quiescent and envelop differentiating cysts to protect them from niche signaling, an important role for germline differentiation (Chen et al. 2011). ECs guide differentiating cysts to the posterior end of the germarium, where the germline becomes encapsulated by the follicular epithelium and pinched off from the germarium. (B) The Drosophila testes are a paired tubular organ that consists of somatic and germline cells. Scheme depicting the testis apex somatic cells (hub cells, Hub; cyst stem cells, CySC’s; cyst stem cells lineage, CySC lineage) and germline cells (germline stem cells, GSCs; gonioblast, GB; differentiating spermatocysts and spermatogonia). Attached to the stem cell niche, termed the hub, reside two types of stem cells: GSCs and CySC’s. While the hub is made by a cluster of postmitotic somatic cells, both stem cell types divide in synchrony to produce differentiating germline cysts, each of which is encapsulated by two somatic CySC’s lineage cells. This encapsulation is critical for proper germline differentiation (Leatherman and Dinardo 2010). Similar to the ovarian GSC progeny, the germline gonioblast undergoes four rounds of incomplete cytokinesis to produce 16 primary spermatocytes in a cyst, eventually generating 64 sperm cells. (C) The relative expression levels of miRNA let-7 in female and male carcasses, ovaries, and testes show that let-7 miRNA is sex biased and expressed at the higher levels in testes (see also Table S17). (D, D′, E, E′) Localization of let-7-C miRNAs in the ovaries and testes, detected via membrane GFP and nuclear lacZ expressed under the control of the let-7-C promoter (let-7-CGK1–Gal4/UAS–CD8-GFP::nuc–lacZ). In the ovary, let-7-C is expressed in the somatic cells of the germarium, CpCs and ECs (D). (D′) More ECs express let-7-C, shown by the presence of lacZ (β-Gal, green) when adult flies were subject to a heat shock for 1 hr prior to dissection. (E and E′) In the testis, let-7-C is broadly expressed in all somatic cells, CySC’s and their lineage and the hub cells (let-7-CGK1-Gal4/UAS-CD8-GFP::nuc-lacZ). (F and F′) Control let-7-C rescue germaria (P{W8, let-7-C}/+; let-7-CGK1/KO1) show typical numbers of germline SSCs and developing cysts (marked by the spectrosome and fusome marker Adducin, Add, red) as well as somatic ECs (marked by Traffic jam, Tj, green). The GSC niche (marked by Lamin C, LamC, red) is outlined by yellow dashed lines. (G and G′) Somatic cell ∆let-7 clones (Ubi–GFP, FRT40A/ FRT40A, let-7 miR-125; bab1–Gal4, UAS–Flp/P{W8, let-7-CΔlet-7}) are marked by the absence of GFP and outlined by white dashed lines. Upon let-7 depletion in the soma of the germarium, the number of somatic ECs and germline SSCs increases, while the number of fusome-containing cysts decreases. The magnification is the same in F and G. (H) The testicular apex of a control let-7-C rescue (P{W8, let-7-C}; let-7-CGK1/KO1) displays typical numbers of germline CBs and cysts (marked by spectrosome and fusome marker Add, red) and the CySC lineage cells (marked by Tj, green); yellow dashed lines outline the hub (marked by Fasciclin III, Fas3, red). (J) In Δlet-7 mutant testis (let-7-CGK1/KO1; P{W8, let-7-CΔlet-7}/+) the CySC lineage cells cluster in larger groups and express the ovarian follicular epithelium marker, Fas3. These clusters (outlined by white dashed lines) can be found at the apex or the side of the testicular tube. (I) Percentages of mutant testes containing large ≥5 or >10 somatic cell clusters in comparison to control let-7-C Rescue. Ten to 20 testes were analyzed for each genotype. Red: LamC + Add in D, D′, F, F′, G, G′ ; Fas3 + Add in E, H, J, J′. Green: GFP + β-Gal in D and E; GFP in G, G′; Tj in F, F′, H, J, J′. White: GFP + β-Gal in E′. Blue: DAPI in D–H, G–J′. D, E + E′, H, J + J′ are single confocal sections while D′, F + F′, and G + G′ are projections of several z-stacks.
Mentions: We analyzed let-7-C expression pattern in the adult Drosophila gonads using Gal4 under the control of the intrinsic let-7-C promoter. In the germarium, let-7-C is expressed in some of the somatic escort (ECs) and cap cells (CpCs; Figure 3, A, B, and D). The number of let-7-C-expressing cells and the activity of the let-7-C promoter per se fluctuate in CpCs and ECs in different germaria and within the same germarium (compare Figure 3, D and D′), demonstrating that the expression pattern of let-7-C is highly dynamic.

Bottom Line: Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation.Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads.This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 fagegalt@cshl.edu halyna.shcherbata@mpibpc.mpg.de.

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Related in: MedlinePlus