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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.


Loss of Rbp4 increases cell death. (A) Rbp4 sequence coverage from mass spectrometry (red). (B–E) Brightfield dorsal and lateral (B′–E′) view of control MO (B), control MO + p53 MO (C), rbp4 MO (D) and rbp4 MO + p53 MO (E) embryos. (F) Quantification of TUNEL after rbp4 MO and mRNA rescue. (G–J, L–O, R–S) Dorsal view of TUNEL (green) and propidium iodide (red). (G–J) TUNEL staining in control MO + p53 MO (G), control MO + rbp4 mRNA+ p53 MO (H), rbp4 MO+ p53 MO (I), rbp4 MO + rbp4 mRNA + p53 MO (J). (K) Method for RA rescue of rbp4 MO. (L–O) TUNEL staining in control MO + p53 MO + DMSO (L) control MO + p53 MO + RA (M), rbp4 MO+ p53 MO + DMSO (N), rbp4 MO + p53 MO + RA (O). (P) Quantification of TUNEL in RA rescue of rbp4 MO. (Q) Method of injection of Rbp4 inhibitor, A1120. (R‐S) TUNEL staining in DMSO (R) or A1120 (S) injected embryos (T) Quantification of TUNEL in A1120 or DMSO injected embryos. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.
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dneu22300-fig-0005: Loss of Rbp4 increases cell death. (A) Rbp4 sequence coverage from mass spectrometry (red). (B–E) Brightfield dorsal and lateral (B′–E′) view of control MO (B), control MO + p53 MO (C), rbp4 MO (D) and rbp4 MO + p53 MO (E) embryos. (F) Quantification of TUNEL after rbp4 MO and mRNA rescue. (G–J, L–O, R–S) Dorsal view of TUNEL (green) and propidium iodide (red). (G–J) TUNEL staining in control MO + p53 MO (G), control MO + rbp4 mRNA+ p53 MO (H), rbp4 MO+ p53 MO (I), rbp4 MO + rbp4 mRNA + p53 MO (J). (K) Method for RA rescue of rbp4 MO. (L–O) TUNEL staining in control MO + p53 MO + DMSO (L) control MO + p53 MO + RA (M), rbp4 MO+ p53 MO + DMSO (N), rbp4 MO + p53 MO + RA (O). (P) Quantification of TUNEL in RA rescue of rbp4 MO. (Q) Method of injection of Rbp4 inhibitor, A1120. (R‐S) TUNEL staining in DMSO (R) or A1120 (S) injected embryos (T) Quantification of TUNEL in A1120 or DMSO injected embryos. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.

Mentions: The ability of RA to prevent cell death is consistent with a role for Rbp4 in normal neuroepithelial cell survival. Rbp4 was identified by our mass spectrometry analysis with ∼40% coverage [Fig. 5(A)] suggesting that it may be a CSF signaling factor. Rbp4 is highly expressed in the yolk syncytial layer and to lower levels in the epidermis surrounding the neural tube (Tingaud‐Sequeira et al., 2006; Li et al., 2007) [Supporting Information Fig. 6]. We hypothesized that Rbp4 is bound to retinol in the CSF, promoting RA synthesis in the neuroepithelium and therefore cell survival. To test this, we used a previously published morpholino (modified antisense oligonucleotide; MO), which targets the exon 2/intron 3 splice site of rbp4 (Li et al., 2007) and asked whether inhibition of rbp4 increased cell death. While MO technology is useful, stringent controls must be performed to ascertain specificity. As will be described in the following sections, we performed such controls, including rescue of a MO phenotype with injected zebrafish mRNA or human protein in addition to use of chemical agonists and inhibitors, that together solidify the conclusions we are able to draw.


Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway
Loss of Rbp4 increases cell death. (A) Rbp4 sequence coverage from mass spectrometry (red). (B–E) Brightfield dorsal and lateral (B′–E′) view of control MO (B), control MO + p53 MO (C), rbp4 MO (D) and rbp4 MO + p53 MO (E) embryos. (F) Quantification of TUNEL after rbp4 MO and mRNA rescue. (G–J, L–O, R–S) Dorsal view of TUNEL (green) and propidium iodide (red). (G–J) TUNEL staining in control MO + p53 MO (G), control MO + rbp4 mRNA+ p53 MO (H), rbp4 MO+ p53 MO (I), rbp4 MO + rbp4 mRNA + p53 MO (J). (K) Method for RA rescue of rbp4 MO. (L–O) TUNEL staining in control MO + p53 MO + DMSO (L) control MO + p53 MO + RA (M), rbp4 MO+ p53 MO + DMSO (N), rbp4 MO + p53 MO + RA (O). (P) Quantification of TUNEL in RA rescue of rbp4 MO. (Q) Method of injection of Rbp4 inhibitor, A1120. (R‐S) TUNEL staining in DMSO (R) or A1120 (S) injected embryos (T) Quantification of TUNEL in A1120 or DMSO injected embryos. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.
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dneu22300-fig-0005: Loss of Rbp4 increases cell death. (A) Rbp4 sequence coverage from mass spectrometry (red). (B–E) Brightfield dorsal and lateral (B′–E′) view of control MO (B), control MO + p53 MO (C), rbp4 MO (D) and rbp4 MO + p53 MO (E) embryos. (F) Quantification of TUNEL after rbp4 MO and mRNA rescue. (G–J, L–O, R–S) Dorsal view of TUNEL (green) and propidium iodide (red). (G–J) TUNEL staining in control MO + p53 MO (G), control MO + rbp4 mRNA+ p53 MO (H), rbp4 MO+ p53 MO (I), rbp4 MO + rbp4 mRNA + p53 MO (J). (K) Method for RA rescue of rbp4 MO. (L–O) TUNEL staining in control MO + p53 MO + DMSO (L) control MO + p53 MO + RA (M), rbp4 MO+ p53 MO + DMSO (N), rbp4 MO + p53 MO + RA (O). (P) Quantification of TUNEL in RA rescue of rbp4 MO. (Q) Method of injection of Rbp4 inhibitor, A1120. (R‐S) TUNEL staining in DMSO (R) or A1120 (S) injected embryos (T) Quantification of TUNEL in A1120 or DMSO injected embryos. Data represented as mean ± SEM. F = forebrain, M = midbrain, H = hindbrain. Scale bars = 50 μm.
Mentions: The ability of RA to prevent cell death is consistent with a role for Rbp4 in normal neuroepithelial cell survival. Rbp4 was identified by our mass spectrometry analysis with ∼40% coverage [Fig. 5(A)] suggesting that it may be a CSF signaling factor. Rbp4 is highly expressed in the yolk syncytial layer and to lower levels in the epidermis surrounding the neural tube (Tingaud‐Sequeira et al., 2006; Li et al., 2007) [Supporting Information Fig. 6]. We hypothesized that Rbp4 is bound to retinol in the CSF, promoting RA synthesis in the neuroepithelium and therefore cell survival. To test this, we used a previously published morpholino (modified antisense oligonucleotide; MO), which targets the exon 2/intron 3 splice site of rbp4 (Li et al., 2007) and asked whether inhibition of rbp4 increased cell death. While MO technology is useful, stringent controls must be performed to ascertain specificity. As will be described in the following sections, we performed such controls, including rescue of a MO phenotype with injected zebrafish mRNA or human protein in addition to use of chemical agonists and inhibitors, that together solidify the conclusions we are able to draw.

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.