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GPCRs Direct Germline Development and Somatic Gonad Function in Planarians.

Saberi A, Jamal A, Beets I, Schoofs L, Newmark PA - PLoS Biol. (2016)

Bottom Line: Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity.We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians.However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

No MeSH data available.


Related in: MedlinePlus

NPY-8 targets npyr-1+ neuroendocrine cells in the CNS.(A) Schematic of receptor-activation assay performed in CHO/mtAEQ/G16 cells. Candidate GPCRs were expressed transiently in a cell line that enables visualization of calcium mobilization upon receptor activation. (B) Normalized activation response of CHO cells expressing NPYR-1 or control receptors, challenged with NPY-8 or control peptides. NPY-8 specifically activates NPYR-1, while two other NPY receptors (NPYR-7 and NPYR-8) are not activated upon NPY-8 treatment. Scrambled NPY-8 was used as a negative control. Calcium responses were normalized to the total calcium response after addition of 0.1% Triton X-100. ATP, which activates an endogenous CHO receptor, was used to test the functionality of the assay. Peptides and ATP were tested at 10 and 1 μM, respectively. (C) Concentration-response curves for the activation of NPYR-1 by NPY-8 and control peptides. Data are shown as a percentage of the highest normalized response of the concentration series. NPY-8 activates NPYR-1 at EC50 = 36.7 nM (blue). Closely related NPY-1 fails to activate NPYR-1 (purple). Empty pcDNA3.1 vector and NPYR-7 were used as negative controls. Error bars in B and C represent standard error of the mean (SEM) (n ≥ 4). The underlying data for receptor assays can be found in S4 Data. (D) Colorimetric ISH showing expression of npyr-1 in a subset of cells in the brain and along the ventral nerve cords. Inset shows the brain region at higher magnification. (E–G) Double-FISH labeling npyr-1 (red) and other neural markers (green). npyr-1+ cells express neuroendocrine cell marker pc2 (E, 28/30 express pc2) but not the cholinergic neuronal marker ChAT (F, 0/30 express ChAT). npy-8 and npyr-1 are expressed in distinct populations of cells (G, 0/30 npyr-1 cells express npy-8 and 0/30 npy-8 cells express npyr-1). DAPI (grey) labels nuclei. Scale bars are 1 mm in D and 50 μm in E–G.
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pbio.1002457.g003: NPY-8 targets npyr-1+ neuroendocrine cells in the CNS.(A) Schematic of receptor-activation assay performed in CHO/mtAEQ/G16 cells. Candidate GPCRs were expressed transiently in a cell line that enables visualization of calcium mobilization upon receptor activation. (B) Normalized activation response of CHO cells expressing NPYR-1 or control receptors, challenged with NPY-8 or control peptides. NPY-8 specifically activates NPYR-1, while two other NPY receptors (NPYR-7 and NPYR-8) are not activated upon NPY-8 treatment. Scrambled NPY-8 was used as a negative control. Calcium responses were normalized to the total calcium response after addition of 0.1% Triton X-100. ATP, which activates an endogenous CHO receptor, was used to test the functionality of the assay. Peptides and ATP were tested at 10 and 1 μM, respectively. (C) Concentration-response curves for the activation of NPYR-1 by NPY-8 and control peptides. Data are shown as a percentage of the highest normalized response of the concentration series. NPY-8 activates NPYR-1 at EC50 = 36.7 nM (blue). Closely related NPY-1 fails to activate NPYR-1 (purple). Empty pcDNA3.1 vector and NPYR-7 were used as negative controls. Error bars in B and C represent standard error of the mean (SEM) (n ≥ 4). The underlying data for receptor assays can be found in S4 Data. (D) Colorimetric ISH showing expression of npyr-1 in a subset of cells in the brain and along the ventral nerve cords. Inset shows the brain region at higher magnification. (E–G) Double-FISH labeling npyr-1 (red) and other neural markers (green). npyr-1+ cells express neuroendocrine cell marker pc2 (E, 28/30 express pc2) but not the cholinergic neuronal marker ChAT (F, 0/30 express ChAT). npy-8 and npyr-1 are expressed in distinct populations of cells (G, 0/30 npyr-1 cells express npy-8 and 0/30 npy-8 cells express npyr-1). DAPI (grey) labels nuclei. Scale bars are 1 mm in D and 50 μm in E–G.

Mentions: To test whether NPY-8 is able to functionally activate NPYR-1, we performed a cell-based receptor-activation assay. We individually expressed three NPY receptors encoded by the npyr-1, npyr-7, and npyr-8 genes in CHO cells co-expressing the promiscuous Gα16 subunit and mitochondrially targeted apoaequorin (CHO/mtAEQ/G16) [43], which enable sensitive monitoring of intracellular calcium responses to exogenous ligands. We then assayed receptor activation after addition of various concentrations of synthetic NPY-8, as well as a closely related family member, NPY-1, and scrambled NPY-8 as controls (Fig 3A). NPYR-1 was activated by NPY-8, but not by the control ligands; by contrast, cells expressing NPYR-7 or NPYR-8, or transfected with an empty vector were not activated (Fig 3B and 3C). Moreover, concentration-response assays showed that NPY-8 activates NPYR-1 at nanomolar concentrations, with an EC50 value of 36.7 nM (Fig 3C). Taken together, our results suggest that NPYR-1 is the cognate receptor for NPY-8.


GPCRs Direct Germline Development and Somatic Gonad Function in Planarians.

Saberi A, Jamal A, Beets I, Schoofs L, Newmark PA - PLoS Biol. (2016)

NPY-8 targets npyr-1+ neuroendocrine cells in the CNS.(A) Schematic of receptor-activation assay performed in CHO/mtAEQ/G16 cells. Candidate GPCRs were expressed transiently in a cell line that enables visualization of calcium mobilization upon receptor activation. (B) Normalized activation response of CHO cells expressing NPYR-1 or control receptors, challenged with NPY-8 or control peptides. NPY-8 specifically activates NPYR-1, while two other NPY receptors (NPYR-7 and NPYR-8) are not activated upon NPY-8 treatment. Scrambled NPY-8 was used as a negative control. Calcium responses were normalized to the total calcium response after addition of 0.1% Triton X-100. ATP, which activates an endogenous CHO receptor, was used to test the functionality of the assay. Peptides and ATP were tested at 10 and 1 μM, respectively. (C) Concentration-response curves for the activation of NPYR-1 by NPY-8 and control peptides. Data are shown as a percentage of the highest normalized response of the concentration series. NPY-8 activates NPYR-1 at EC50 = 36.7 nM (blue). Closely related NPY-1 fails to activate NPYR-1 (purple). Empty pcDNA3.1 vector and NPYR-7 were used as negative controls. Error bars in B and C represent standard error of the mean (SEM) (n ≥ 4). The underlying data for receptor assays can be found in S4 Data. (D) Colorimetric ISH showing expression of npyr-1 in a subset of cells in the brain and along the ventral nerve cords. Inset shows the brain region at higher magnification. (E–G) Double-FISH labeling npyr-1 (red) and other neural markers (green). npyr-1+ cells express neuroendocrine cell marker pc2 (E, 28/30 express pc2) but not the cholinergic neuronal marker ChAT (F, 0/30 express ChAT). npy-8 and npyr-1 are expressed in distinct populations of cells (G, 0/30 npyr-1 cells express npy-8 and 0/30 npy-8 cells express npyr-1). DAPI (grey) labels nuclei. Scale bars are 1 mm in D and 50 μm in E–G.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4862687&req=5

pbio.1002457.g003: NPY-8 targets npyr-1+ neuroendocrine cells in the CNS.(A) Schematic of receptor-activation assay performed in CHO/mtAEQ/G16 cells. Candidate GPCRs were expressed transiently in a cell line that enables visualization of calcium mobilization upon receptor activation. (B) Normalized activation response of CHO cells expressing NPYR-1 or control receptors, challenged with NPY-8 or control peptides. NPY-8 specifically activates NPYR-1, while two other NPY receptors (NPYR-7 and NPYR-8) are not activated upon NPY-8 treatment. Scrambled NPY-8 was used as a negative control. Calcium responses were normalized to the total calcium response after addition of 0.1% Triton X-100. ATP, which activates an endogenous CHO receptor, was used to test the functionality of the assay. Peptides and ATP were tested at 10 and 1 μM, respectively. (C) Concentration-response curves for the activation of NPYR-1 by NPY-8 and control peptides. Data are shown as a percentage of the highest normalized response of the concentration series. NPY-8 activates NPYR-1 at EC50 = 36.7 nM (blue). Closely related NPY-1 fails to activate NPYR-1 (purple). Empty pcDNA3.1 vector and NPYR-7 were used as negative controls. Error bars in B and C represent standard error of the mean (SEM) (n ≥ 4). The underlying data for receptor assays can be found in S4 Data. (D) Colorimetric ISH showing expression of npyr-1 in a subset of cells in the brain and along the ventral nerve cords. Inset shows the brain region at higher magnification. (E–G) Double-FISH labeling npyr-1 (red) and other neural markers (green). npyr-1+ cells express neuroendocrine cell marker pc2 (E, 28/30 express pc2) but not the cholinergic neuronal marker ChAT (F, 0/30 express ChAT). npy-8 and npyr-1 are expressed in distinct populations of cells (G, 0/30 npyr-1 cells express npy-8 and 0/30 npy-8 cells express npyr-1). DAPI (grey) labels nuclei. Scale bars are 1 mm in D and 50 μm in E–G.
Mentions: To test whether NPY-8 is able to functionally activate NPYR-1, we performed a cell-based receptor-activation assay. We individually expressed three NPY receptors encoded by the npyr-1, npyr-7, and npyr-8 genes in CHO cells co-expressing the promiscuous Gα16 subunit and mitochondrially targeted apoaequorin (CHO/mtAEQ/G16) [43], which enable sensitive monitoring of intracellular calcium responses to exogenous ligands. We then assayed receptor activation after addition of various concentrations of synthetic NPY-8, as well as a closely related family member, NPY-1, and scrambled NPY-8 as controls (Fig 3A). NPYR-1 was activated by NPY-8, but not by the control ligands; by contrast, cells expressing NPYR-7 or NPYR-8, or transfected with an empty vector were not activated (Fig 3B and 3C). Moreover, concentration-response assays showed that NPY-8 activates NPYR-1 at nanomolar concentrations, with an EC50 value of 36.7 nM (Fig 3C). Taken together, our results suggest that NPYR-1 is the cognate receptor for NPY-8.

Bottom Line: Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity.We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians.However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

No MeSH data available.


Related in: MedlinePlus