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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.


Related in: MedlinePlus

CSF promotes cell survival and tail extension. (A) Experimental design. Drainage/puncture (*) occurred every 2 hours from 22 to 36 hpf. Embryos were assayed (A) at 36 hpf. (B–D) Brightfield lateral view of unpunctured (B), punctured (C), and drained (D) embryos. (B′–D′) Whole embryo phenotype. (E) Percent of embryos with curved tails. (F–H) Dorsal view of PH3 (green) and (J–L) TUNEL (green). Propidium iodide in red. (F‐H; J‐L) white box indicates region quantified. (J′,K′L′) Magnified image of region indicated by white box in J,K,L. (I,M) Quantification of the fraction of PH3 (I) and TUNEL (M) positive cells. (N–P) Dorsal view of acetylated tubulin (green) or (Q–S) HuC (red) and propidium iodide (blue). (T) Dorsal/ventral sections of neural tube (T1‐3) stained with TUNEL (green), HuC (red) and propidium iodide (blue). (U) Representative dorsal/ventral sections of neural tube shown in T. 1‐3 correspond to T1‐T3. (V) Magnified image of TUNEL and HuC (V1), HuC alone (V2) and TUNEL alone (V3) from dorsal section of diencephalon (T1). (N‐S) projection overlaid on single slice of propidium iodide image, all other images are a single 0.6 μm slice. 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-0001: CSF promotes cell survival and tail extension. (A) Experimental design. Drainage/puncture (*) occurred every 2 hours from 22 to 36 hpf. Embryos were assayed (A) at 36 hpf. (B–D) Brightfield lateral view of unpunctured (B), punctured (C), and drained (D) embryos. (B′–D′) Whole embryo phenotype. (E) Percent of embryos with curved tails. (F–H) Dorsal view of PH3 (green) and (J–L) TUNEL (green). Propidium iodide in red. (F‐H; J‐L) white box indicates region quantified. (J′,K′L′) Magnified image of region indicated by white box in J,K,L. (I,M) Quantification of the fraction of PH3 (I) and TUNEL (M) positive cells. (N–P) Dorsal view of acetylated tubulin (green) or (Q–S) HuC (red) and propidium iodide (blue). (T) Dorsal/ventral sections of neural tube (T1‐3) stained with TUNEL (green), HuC (red) and propidium iodide (blue). (U) Representative dorsal/ventral sections of neural tube shown in T. 1‐3 correspond to T1‐T3. (V) Magnified image of TUNEL and HuC (V1), HuC alone (V2) and TUNEL alone (V3) from dorsal section of diencephalon (T1). (N‐S) projection overlaid on single slice of propidium iodide image, all other images are a single 0.6 μm slice. Data represented as mean ± SEM. F= forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.

Mentions: Manual drainage technique was performed as previously described (Chang and Sive, 2012). Briefly, a micropipette needle was inserted through the roof plate of the hindbrain ventricle and was positioned either in the hindbrain, midbrain, or forebrain ventricle. CSF was removed from all three brain ventricles, using an Eppendorf CellTram oil microinjector apparatus, every one to two hours from 22–36 hpf (referred to as drained; six times total, [Fig. 1 (A)]). After each drain, the needle was removed and embryos stored at 32°C to speed up development in order to decrease the amount of experimental time required to examine a 14 hour developmental time window. As a control, the micropipette needle was inserted into embryos without removing any CSF (referred to as punctured). Unpunctured and punctured embryos were also stored at 32°C from 22–36 hpf. To introduce factors into drained embryos, 1–2 nL of a factor was injected every 2 hours from 30–36 hpf (three times total, [Fig. 3(A)]) into the brain ventricles as described previously (Gutzman and Sive, 2009). Factors used include: E10.5 mouse CSF (frozen), physiological saline/artificial CSF (118 mM NaCl, 2 mM KCl, 10 mM MgCl2, 10 mM HEPES, 10 mM glucose), Caspase 3 Inhibitor I (Millipore; 500uM), Insulin like growth factor 2 recombinant human (IGF2, US Biologicals, 25ng/mL), Fibroblast growth factor 2 (FGF2, Sigma, 300 μg/mL), all‐trans RA (Sigma, 10−8M), all‐trans retinol (Sigma, 348 nM), recombinant human RBP4 (R&D systems, 2 ng/μL), A1120 (Sigma 30 × 10−8M), Citral (Sigma 250 μM), 4‐Diethylaminobenzaldehyde (DEAB, Sigma, 10 μM). DMSO diluted 1:10‐1:1000 in E3 or E3 alone were used as negative control injections.


Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway
CSF promotes cell survival and tail extension. (A) Experimental design. Drainage/puncture (*) occurred every 2 hours from 22 to 36 hpf. Embryos were assayed (A) at 36 hpf. (B–D) Brightfield lateral view of unpunctured (B), punctured (C), and drained (D) embryos. (B′–D′) Whole embryo phenotype. (E) Percent of embryos with curved tails. (F–H) Dorsal view of PH3 (green) and (J–L) TUNEL (green). Propidium iodide in red. (F‐H; J‐L) white box indicates region quantified. (J′,K′L′) Magnified image of region indicated by white box in J,K,L. (I,M) Quantification of the fraction of PH3 (I) and TUNEL (M) positive cells. (N–P) Dorsal view of acetylated tubulin (green) or (Q–S) HuC (red) and propidium iodide (blue). (T) Dorsal/ventral sections of neural tube (T1‐3) stained with TUNEL (green), HuC (red) and propidium iodide (blue). (U) Representative dorsal/ventral sections of neural tube shown in T. 1‐3 correspond to T1‐T3. (V) Magnified image of TUNEL and HuC (V1), HuC alone (V2) and TUNEL alone (V3) from dorsal section of diencephalon (T1). (N‐S) projection overlaid on single slice of propidium iodide image, all other images are a single 0.6 μm slice. 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-0001: CSF promotes cell survival and tail extension. (A) Experimental design. Drainage/puncture (*) occurred every 2 hours from 22 to 36 hpf. Embryos were assayed (A) at 36 hpf. (B–D) Brightfield lateral view of unpunctured (B), punctured (C), and drained (D) embryos. (B′–D′) Whole embryo phenotype. (E) Percent of embryos with curved tails. (F–H) Dorsal view of PH3 (green) and (J–L) TUNEL (green). Propidium iodide in red. (F‐H; J‐L) white box indicates region quantified. (J′,K′L′) Magnified image of region indicated by white box in J,K,L. (I,M) Quantification of the fraction of PH3 (I) and TUNEL (M) positive cells. (N–P) Dorsal view of acetylated tubulin (green) or (Q–S) HuC (red) and propidium iodide (blue). (T) Dorsal/ventral sections of neural tube (T1‐3) stained with TUNEL (green), HuC (red) and propidium iodide (blue). (U) Representative dorsal/ventral sections of neural tube shown in T. 1‐3 correspond to T1‐T3. (V) Magnified image of TUNEL and HuC (V1), HuC alone (V2) and TUNEL alone (V3) from dorsal section of diencephalon (T1). (N‐S) projection overlaid on single slice of propidium iodide image, all other images are a single 0.6 μm slice. Data represented as mean ± SEM. F= forebrain, M = midbrain. UP = unpunctured, P = punctured, D = drained. Scale bars = 50 μm.
Mentions: Manual drainage technique was performed as previously described (Chang and Sive, 2012). Briefly, a micropipette needle was inserted through the roof plate of the hindbrain ventricle and was positioned either in the hindbrain, midbrain, or forebrain ventricle. CSF was removed from all three brain ventricles, using an Eppendorf CellTram oil microinjector apparatus, every one to two hours from 22–36 hpf (referred to as drained; six times total, [Fig. 1 (A)]). After each drain, the needle was removed and embryos stored at 32°C to speed up development in order to decrease the amount of experimental time required to examine a 14 hour developmental time window. As a control, the micropipette needle was inserted into embryos without removing any CSF (referred to as punctured). Unpunctured and punctured embryos were also stored at 32°C from 22–36 hpf. To introduce factors into drained embryos, 1–2 nL of a factor was injected every 2 hours from 30–36 hpf (three times total, [Fig. 3(A)]) into the brain ventricles as described previously (Gutzman and Sive, 2009). Factors used include: E10.5 mouse CSF (frozen), physiological saline/artificial CSF (118 mM NaCl, 2 mM KCl, 10 mM MgCl2, 10 mM HEPES, 10 mM glucose), Caspase 3 Inhibitor I (Millipore; 500uM), Insulin like growth factor 2 recombinant human (IGF2, US Biologicals, 25ng/mL), Fibroblast growth factor 2 (FGF2, Sigma, 300 μg/mL), all‐trans RA (Sigma, 10−8M), all‐trans retinol (Sigma, 348 nM), recombinant human RBP4 (R&D systems, 2 ng/μL), A1120 (Sigma 30 × 10−8M), Citral (Sigma 250 μM), 4‐Diethylaminobenzaldehyde (DEAB, Sigma, 10 μM). DMSO diluted 1:10‐1:1000 in E3 or E3 alone were used as negative control injections.

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