Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Abstract

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules 1,2 . In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles [3][4][5] . In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf) 6 . Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development 7 . eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development 8,9 . Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium [10][11][12][13][14][15][16][17][18][19][20] . Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation [21][22][23][24] . However, the complex functions of the eCSF are largely unknown.We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development. Video LinkThe video component of this article can be found at

Physiology and molecular biology of barrier mechanisms in the fetal and neonatal brain

“…These have the considerable advantages of accessibility at very early stages of brain development. Zebrafish have the so‐far‐unique advantage of being transparent so various processes can be visualized real‐time with time‐lapse microscopy (Chang & Sive, ; Umans et al . ).…”

Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

“…We began with the hypothesis that CSF is required for proper embryonic brain development. To address this, we used an in vivo technique to manually drain CSF from embryonic zebrafish brain ventricles generating an CSF‐depleted embryo (Chang and Sive, ). We drained CSF from 22 hours post fertilization (hpf), when CSF first begins to fill zebrafish embryonic brain ventricles, to 36 hpf spanning a period during which there is a significant amount of cell proliferation, neuronal differentiation, and axonal projection necessary for proper brain morphogenesis and development (Kimmel and Westerfield, ; Lowery and Sive, ).…”

“…We drained CSF from 22 hours post fertilization (hpf), when CSF first begins to fill zebrafish embryonic brain ventricles, to 36 hpf spanning a period during which there is a significant amount of cell proliferation, neuronal differentiation, and axonal projection necessary for proper brain morphogenesis and development (Kimmel and Westerfield, ; Lowery and Sive, ). CSF can be fully replenished after 2–3 hours (Chang and Sive, ), therefore, we drained CSF every two hours from 22–36 hpf [Fig. (A)].…”

“…Manual drainage technique was performed as previously described (Chang and Sive, ). 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.…”

“…The zebrafish is an ideal system to define the mechanisms underlying CSF function during brain development (Lowery and Sive, ; Gutzman and Sive, ; Chang et al, ). Since embryonic development occurs externally CSF withdrawal and factor replacement can be performed in living embryos (Chang and Sive, ). This study is the first characterization of CSF function in embryonic zebrafish after chronic removal.…”