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Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster.

Kis V, Barti B, Lippai M, Sass M - PLoS ONE (2015)

Bottom Line: Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described.Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry.To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary.

ABSTRACT
Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

No MeSH data available.


Related in: MedlinePlus

Light microscopic localisation of the Drosophila fatty acid binding protein (Dfabp) in third instar larvae.(A) Western blot performed on total protein extracts from dfabp3252 homozygous mutant and wild type larvae. The Dfabp antibody labels a single band at 14kDa in the wild type sample, while no labeling is observed in samples from mutants. Immunohistochemistry on wild type (B), and on dfabp RNAi (C) third instar larval brains using the Dfabp antibody (green). Nuclei are stained with DAPI (blue). Note the absence of staining in RNAi silenced compared to wild type animals. (D) Higher magnification image of the dorsomedial part of the central brain. The Dfabp antibody reveals a thin network, between neuroblasts (asterisks) and their daughter cells. Two Dfabp-positive (arrowheads), and one unlabeled soma (arrow) are visible at the brain surface. (E) Double labeling against Dfabp (green) and the glial-specific protein Repo (red). Note that Dfabp is present in the cytosol and in the nucleus of glial cells (arrows). (F) Double labeling for cortex glia-GFP (green) and Dfabp (red). Perineurial cells (arrow) are double negative for Dfabp and GFP. Subperineurial cells (arrowhead) are positive for Dfabp and negative for GFP. Cortex glial cells (double arrow) are double positive for Dfabp and GFP. Scalebar: B, C:100 μm; D, E, F, F’: 10 μm.
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pone.0131250.g005: Light microscopic localisation of the Drosophila fatty acid binding protein (Dfabp) in third instar larvae.(A) Western blot performed on total protein extracts from dfabp3252 homozygous mutant and wild type larvae. The Dfabp antibody labels a single band at 14kDa in the wild type sample, while no labeling is observed in samples from mutants. Immunohistochemistry on wild type (B), and on dfabp RNAi (C) third instar larval brains using the Dfabp antibody (green). Nuclei are stained with DAPI (blue). Note the absence of staining in RNAi silenced compared to wild type animals. (D) Higher magnification image of the dorsomedial part of the central brain. The Dfabp antibody reveals a thin network, between neuroblasts (asterisks) and their daughter cells. Two Dfabp-positive (arrowheads), and one unlabeled soma (arrow) are visible at the brain surface. (E) Double labeling against Dfabp (green) and the glial-specific protein Repo (red). Note that Dfabp is present in the cytosol and in the nucleus of glial cells (arrows). (F) Double labeling for cortex glia-GFP (green) and Dfabp (red). Perineurial cells (arrow) are double negative for Dfabp and GFP. Subperineurial cells (arrowhead) are positive for Dfabp and negative for GFP. Cortex glial cells (double arrow) are double positive for Dfabp and GFP. Scalebar: B, C:100 μm; D, E, F, F’: 10 μm.

Mentions: Next, we wanted to find molecular markers that would be specific to LD accumulating superficial cortex glial cells. With this objective in mind, we performed a literature based screen for lipid metabolism-related genes expressed in the Drosophila CNS. We found that the Drosophila fatty acid binding protein (dfabp,CG6783), an orthologue of the mammalian fatty acid binding protein 7 (FABP7/brain lipid binding protein, BLBP) is preferentially expressed in glial cells of the embryonic nervous system, based on in situ hybridization, and microarray analysis [34–37]. We were interested in whether or not this protein is indeed expressed in third instar larval glial cells. To test this, we raised polyclonal antisera against the third exon of dfabp. On Western blots from wild type animals a single band (14kDa) was detected corresponding the predicted molecular mass of dfabp, while no labeling was observed in samples from larvae homozygous for dfabpEP3252, a strong hypomorf mutation of dfabp (Fig 5A). Since the dfabpEP3252 mutation causes early larval lethality, we validated our antibody for immunohistochemistry using transgenic flies in which dfabp was silenced with a microRNA based RNAi construct specifically expressed in all glial cells using repo-Gal4. The antibody strongly labelled the brains of wild type but not the dfabp RNAi silenced brains, which confirms the specificity of the anti-Dfabp antibody (Fig 5B and 5C).


Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster.

Kis V, Barti B, Lippai M, Sass M - PLoS ONE (2015)

Light microscopic localisation of the Drosophila fatty acid binding protein (Dfabp) in third instar larvae.(A) Western blot performed on total protein extracts from dfabp3252 homozygous mutant and wild type larvae. The Dfabp antibody labels a single band at 14kDa in the wild type sample, while no labeling is observed in samples from mutants. Immunohistochemistry on wild type (B), and on dfabp RNAi (C) third instar larval brains using the Dfabp antibody (green). Nuclei are stained with DAPI (blue). Note the absence of staining in RNAi silenced compared to wild type animals. (D) Higher magnification image of the dorsomedial part of the central brain. The Dfabp antibody reveals a thin network, between neuroblasts (asterisks) and their daughter cells. Two Dfabp-positive (arrowheads), and one unlabeled soma (arrow) are visible at the brain surface. (E) Double labeling against Dfabp (green) and the glial-specific protein Repo (red). Note that Dfabp is present in the cytosol and in the nucleus of glial cells (arrows). (F) Double labeling for cortex glia-GFP (green) and Dfabp (red). Perineurial cells (arrow) are double negative for Dfabp and GFP. Subperineurial cells (arrowhead) are positive for Dfabp and negative for GFP. Cortex glial cells (double arrow) are double positive for Dfabp and GFP. Scalebar: B, C:100 μm; D, E, F, F’: 10 μm.
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Related In: Results  -  Collection

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pone.0131250.g005: Light microscopic localisation of the Drosophila fatty acid binding protein (Dfabp) in third instar larvae.(A) Western blot performed on total protein extracts from dfabp3252 homozygous mutant and wild type larvae. The Dfabp antibody labels a single band at 14kDa in the wild type sample, while no labeling is observed in samples from mutants. Immunohistochemistry on wild type (B), and on dfabp RNAi (C) third instar larval brains using the Dfabp antibody (green). Nuclei are stained with DAPI (blue). Note the absence of staining in RNAi silenced compared to wild type animals. (D) Higher magnification image of the dorsomedial part of the central brain. The Dfabp antibody reveals a thin network, between neuroblasts (asterisks) and their daughter cells. Two Dfabp-positive (arrowheads), and one unlabeled soma (arrow) are visible at the brain surface. (E) Double labeling against Dfabp (green) and the glial-specific protein Repo (red). Note that Dfabp is present in the cytosol and in the nucleus of glial cells (arrows). (F) Double labeling for cortex glia-GFP (green) and Dfabp (red). Perineurial cells (arrow) are double negative for Dfabp and GFP. Subperineurial cells (arrowhead) are positive for Dfabp and negative for GFP. Cortex glial cells (double arrow) are double positive for Dfabp and GFP. Scalebar: B, C:100 μm; D, E, F, F’: 10 μm.
Mentions: Next, we wanted to find molecular markers that would be specific to LD accumulating superficial cortex glial cells. With this objective in mind, we performed a literature based screen for lipid metabolism-related genes expressed in the Drosophila CNS. We found that the Drosophila fatty acid binding protein (dfabp,CG6783), an orthologue of the mammalian fatty acid binding protein 7 (FABP7/brain lipid binding protein, BLBP) is preferentially expressed in glial cells of the embryonic nervous system, based on in situ hybridization, and microarray analysis [34–37]. We were interested in whether or not this protein is indeed expressed in third instar larval glial cells. To test this, we raised polyclonal antisera against the third exon of dfabp. On Western blots from wild type animals a single band (14kDa) was detected corresponding the predicted molecular mass of dfabp, while no labeling was observed in samples from larvae homozygous for dfabpEP3252, a strong hypomorf mutation of dfabp (Fig 5A). Since the dfabpEP3252 mutation causes early larval lethality, we validated our antibody for immunohistochemistry using transgenic flies in which dfabp was silenced with a microRNA based RNAi construct specifically expressed in all glial cells using repo-Gal4. The antibody strongly labelled the brains of wild type but not the dfabp RNAi silenced brains, which confirms the specificity of the anti-Dfabp antibody (Fig 5B and 5C).

Bottom Line: Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described.Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry.To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary.

ABSTRACT
Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

No MeSH data available.


Related in: MedlinePlus