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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.


RA prevents neuroepithelial cell death at 36 and 48 hpf. (A–D) Dorsal view of TUNEL (green) and propidum iodide (red) of un‐punctured (A), punctured (B) or drained embryos (C) injected with DMSO and drained embryos injected with RA (D). (E–F) Quantification of cell death at 36 hpf after introduction of FGF2, IGF2, or RA (E) or at 48 hpf after introduction of RA (F). 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-0004: RA prevents neuroepithelial cell death at 36 and 48 hpf. (A–D) Dorsal view of TUNEL (green) and propidum iodide (red) of un‐punctured (A), punctured (B) or drained embryos (C) injected with DMSO and drained embryos injected with RA (D). (E–F) Quantification of cell death at 36 hpf after introduction of FGF2, IGF2, or RA (E) or at 48 hpf after introduction of RA (F). Data represented as mean ± SEM. F = forebrain, M = midbrain, UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.

Mentions: We hypothesized that among the CSF factors identified one or more was required for neuroepithelial cell survival. One interesting candidate was Rbp4 [Supporting Information Table 1], which has also been identified within human, rat, and chick CSF (Zappaterra et al., 2007; Parada et al., 2008). RBP4 is a plasma protein that transports retinol, the precursor for RA. We therefore hypothesized that RA would mediate cell survival through the CSF. To test this, RA or DMSO was injected from 30 to 36 hpf into embryos drained or punctured from 22 to 36 hpf, or unpunctured embryos [Fig. 3(A), Fig. 4(A–D), Supporting Information Fig. 5]. After RA injection into drained brain ventricles, cell death was reduced (n = 16, p < 0.0001) compared with DMSO controls (n = 39) [Fig. 4(C–E)] to levels comparable to punctured or unpunctured embryos [Fig. 4(A, B, E), Supporting Information Fig. 5] (unpunctured + DMSO [n = 41], punctured + DMSO [n = 38]). Exogenous RA injected into un‐punctured or punctured embryos did not change cell death levels compared with DMSO injection demonstrating that RA cannot block “normal” cell death observed during development [Supporting Information Fig. 5]. Again, the curved tail phenotype observed in drained embryos was not prevented by RA injection [Supporting Information Fig. 5] suggesting a separate function or timing of CSF in this phenotype. Similar results were obtained when embryos were assayed at 48 hpf after CSF drainage from 22 to 36 hpf. Levels of cell death were significantly reduced in drained embryos injected with RA embryos (n = 16, p = 0.0031) compared with DMSO (n = 20) [Fig. 4(F)] (unpunctured + DMSO, n = 13; punctured + DMSO, n = 17). These data suggest that the increase in neuroepithelial cell death is first observed at 30 hpf, persists to 48 hpf, and can be reversed by application of exogenous RA.


Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway
RA prevents neuroepithelial cell death at 36 and 48 hpf. (A–D) Dorsal view of TUNEL (green) and propidum iodide (red) of un‐punctured (A), punctured (B) or drained embryos (C) injected with DMSO and drained embryos injected with RA (D). (E–F) Quantification of cell death at 36 hpf after introduction of FGF2, IGF2, or RA (E) or at 48 hpf after introduction of RA (F). 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-0004: RA prevents neuroepithelial cell death at 36 and 48 hpf. (A–D) Dorsal view of TUNEL (green) and propidum iodide (red) of un‐punctured (A), punctured (B) or drained embryos (C) injected with DMSO and drained embryos injected with RA (D). (E–F) Quantification of cell death at 36 hpf after introduction of FGF2, IGF2, or RA (E) or at 48 hpf after introduction of RA (F). Data represented as mean ± SEM. F = forebrain, M = midbrain, UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.
Mentions: We hypothesized that among the CSF factors identified one or more was required for neuroepithelial cell survival. One interesting candidate was Rbp4 [Supporting Information Table 1], which has also been identified within human, rat, and chick CSF (Zappaterra et al., 2007; Parada et al., 2008). RBP4 is a plasma protein that transports retinol, the precursor for RA. We therefore hypothesized that RA would mediate cell survival through the CSF. To test this, RA or DMSO was injected from 30 to 36 hpf into embryos drained or punctured from 22 to 36 hpf, or unpunctured embryos [Fig. 3(A), Fig. 4(A–D), Supporting Information Fig. 5]. After RA injection into drained brain ventricles, cell death was reduced (n = 16, p < 0.0001) compared with DMSO controls (n = 39) [Fig. 4(C–E)] to levels comparable to punctured or unpunctured embryos [Fig. 4(A, B, E), Supporting Information Fig. 5] (unpunctured + DMSO [n = 41], punctured + DMSO [n = 38]). Exogenous RA injected into un‐punctured or punctured embryos did not change cell death levels compared with DMSO injection demonstrating that RA cannot block “normal” cell death observed during development [Supporting Information Fig. 5]. Again, the curved tail phenotype observed in drained embryos was not prevented by RA injection [Supporting Information Fig. 5] suggesting a separate function or timing of CSF in this phenotype. Similar results were obtained when embryos were assayed at 48 hpf after CSF drainage from 22 to 36 hpf. Levels of cell death were significantly reduced in drained embryos injected with RA embryos (n = 16, p = 0.0031) compared with DMSO (n = 20) [Fig. 4(F)] (unpunctured + DMSO, n = 13; punctured + DMSO, n = 17). These data suggest that the increase in neuroepithelial cell death is first observed at 30 hpf, persists to 48 hpf, and can be reversed by application of exogenous RA.

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&#8208;of&#8208;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. &copy; 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75&ndash;92, 2016

No MeSH data available.