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Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

View Article: PubMed Central - PubMed

ABSTRACT

Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss‐of‐function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75–92, 2016

No MeSH data available.


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CSF is required from 25 to 30 hpf, while cell death persists at 48 hpf. (A) Quantification of TUNEL. X axis indicates puncture/drainage period. All assayed at 36 hpf. (B) Quantification of TUNEL at 30 hpf after puncture/drainage from 22 to 30 hpf. (C–E) Brightfield dorsal and lateral (C′–E′) images of 48 hpf embryos after puncture/drainage from 22 to 36 hpf. (F) Quantification of TUNEL at 48 hpf. (G–J) Dorsal view of TUNEL (green) and propidium iodide (red) in 48 hpf embryos after puncture/drainage from either 22–36 hpf (H–I) or 22–30 hpf (J) or unpunctured (G). Data represented as mean ± SEM. F = forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.
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dneu22300-fig-0002: CSF is required from 25 to 30 hpf, while cell death persists at 48 hpf. (A) Quantification of TUNEL. X axis indicates puncture/drainage period. All assayed at 36 hpf. (B) Quantification of TUNEL at 30 hpf after puncture/drainage from 22 to 30 hpf. (C–E) Brightfield dorsal and lateral (C′–E′) images of 48 hpf embryos after puncture/drainage from 22 to 36 hpf. (F) Quantification of TUNEL at 48 hpf. (G–J) Dorsal view of TUNEL (green) and propidium iodide (red) in 48 hpf embryos after puncture/drainage from either 22–36 hpf (H–I) or 22–30 hpf (J) or unpunctured (G). Data represented as mean ± SEM. F = forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.

Mentions: To identify developmental periods where CSF promotes cell survival, we removed fluid for differing lengths of time. Embryos that were drained only once at 22 hpf (n = 10, p = 0.60), or twice from 22–24 hpf (n = 8, p = 0.76) and allowed to recover until 36 hpf did not show increased cell death compared with controls [Fig. 2(A), Supporting Information Fig. 3]. Draining from 22–26 hpf with subsequent recovery until 36 hpf (n = 10, p = 0.15), resulted in a slight increase in cell death [Fig. 2(A), Supporting Information Fig. 3], while removal of CSF from 22–28 (n = 9, p = 0.04) or 22–30 hpf (n = 6, p = 0.0006) with recovery until 36 hpf was associated with significantly increased levels of cell death compared with unpunctured or punctured embryos [Fig. 2(A), Supporting Information Fig. 3]. Furthermore, we observed a significant increase in cell death in embryos drained from 22–30 hpf (n = 8, p = 0.007) and immediately assayed at 30 hpf [Fig. 2(B), Supporting Information Fig. 3]. Together, the data suggest a requirement for CSF from 25 to 30 hpf to promote cell survival during early stages of embryonic brain development.


Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway
CSF is required from 25 to 30 hpf, while cell death persists at 48 hpf. (A) Quantification of TUNEL. X axis indicates puncture/drainage period. All assayed at 36 hpf. (B) Quantification of TUNEL at 30 hpf after puncture/drainage from 22 to 30 hpf. (C–E) Brightfield dorsal and lateral (C′–E′) images of 48 hpf embryos after puncture/drainage from 22 to 36 hpf. (F) Quantification of TUNEL at 48 hpf. (G–J) Dorsal view of TUNEL (green) and propidium iodide (red) in 48 hpf embryos after puncture/drainage from either 22–36 hpf (H–I) or 22–30 hpf (J) or unpunctured (G). Data represented as mean ± SEM. F = forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.
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dneu22300-fig-0002: CSF is required from 25 to 30 hpf, while cell death persists at 48 hpf. (A) Quantification of TUNEL. X axis indicates puncture/drainage period. All assayed at 36 hpf. (B) Quantification of TUNEL at 30 hpf after puncture/drainage from 22 to 30 hpf. (C–E) Brightfield dorsal and lateral (C′–E′) images of 48 hpf embryos after puncture/drainage from 22 to 36 hpf. (F) Quantification of TUNEL at 48 hpf. (G–J) Dorsal view of TUNEL (green) and propidium iodide (red) in 48 hpf embryos after puncture/drainage from either 22–36 hpf (H–I) or 22–30 hpf (J) or unpunctured (G). Data represented as mean ± SEM. F = forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.
Mentions: To identify developmental periods where CSF promotes cell survival, we removed fluid for differing lengths of time. Embryos that were drained only once at 22 hpf (n = 10, p = 0.60), or twice from 22–24 hpf (n = 8, p = 0.76) and allowed to recover until 36 hpf did not show increased cell death compared with controls [Fig. 2(A), Supporting Information Fig. 3]. Draining from 22–26 hpf with subsequent recovery until 36 hpf (n = 10, p = 0.15), resulted in a slight increase in cell death [Fig. 2(A), Supporting Information Fig. 3], while removal of CSF from 22–28 (n = 9, p = 0.04) or 22–30 hpf (n = 6, p = 0.0006) with recovery until 36 hpf was associated with significantly increased levels of cell death compared with unpunctured or punctured embryos [Fig. 2(A), Supporting Information Fig. 3]. Furthermore, we observed a significant increase in cell death in embryos drained from 22–30 hpf (n = 8, p = 0.007) and immediately assayed at 30 hpf [Fig. 2(B), Supporting Information Fig. 3]. Together, the data suggest a requirement for CSF from 25 to 30 hpf to promote cell survival during early stages of embryonic brain development.

View Article: PubMed Central - PubMed

ABSTRACT

Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss‐of‐function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75–92, 2016

No MeSH data available.


Related in: MedlinePlus