Isolation and culture of primary embryonic zebrafish neural tissue

Patel, Bhavika B.; Clark, Kendra L.; Kozik, Emily; Dash, Linkan; Kuhlman, Julie A.; Sakaguchi, Donald S.

doi:10.1016/j.jneumeth.2019.108419

Cited by 10 publications

(9 citation statements)

References 51 publications

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“…3 – 5 ). We next wondered whether Rgs4 is required for Akt activity specifically within the nervous system, we therefore dissociated embryos and generated neural cells enriched cultures 38 from WT and MZrgs4 embryos at 48 hpf to quantify p-Akt/Akt ratio in primary neural cultures. The latter was significantly decreased in MZrgs4 neural extracts in comparison to WT (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…48 hpf WT and MZrgs4 embryos were bleached for 2 min with 0.005% hypochlorite, washed three times with water and anesthetized with 0.03% tricaine before dissection as described in 38 . 35,000 cells per well (24 well plates) were incubated overnight at 29° in zebrafish complete media composed of L-15 (Gibco), supplemented with 2.5 mM glutamax (Fisher Scientific), 15 ng/mL EGF (Fisher Scientific), 10% fetal bovine serum and 5% zebrafish extract (prepared as detailed in 61 ).…”

Section: Methodsmentioning

confidence: 99%

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Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish

Mikdache

Boueid

Spek

et al. 2021

Sci Rep

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The Regulator of G protein signaling 4 (Rgs4) is a member of the RGS proteins superfamily that modulates the activity of G-protein coupled receptors. It is mainly expressed in the nervous system and is linked to several neuronal signaling pathways; however, its role in neural development in vivo remains inconclusive. Here, we generated and characterized a rgs4 loss of function model (MZrgs4) in zebrafish. MZrgs4 embryos showed motility defects and presented reduced head and eye sizes, reflecting defective motoneurons axon outgrowth and a significant decrease in the number of neurons in the central and peripheral nervous system. Forcing the expression of Rgs4 specifically within motoneurons rescued their early defective outgrowth in MZrgs4 embryos, indicating an autonomous role for Rgs4 in motoneurons. We also analyzed the role of Akt, Erk and mechanistic target of rapamycin (mTOR) signaling cascades and showed a requirement for these pathways in motoneurons axon outgrowth and neuronal development. Drawing on pharmacological and rescue experiments in MZrgs4, we provide evidence that Rgs4 facilitates signaling mediated by Akt, Erk and mTOR in order to drive axon outgrowth in motoneurons and regulate neuronal numbers.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish

Mikdache

Boueid

Spek

et al. 2021

Sci Rep

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“…Growth and purity of neuronal cell populations purified using the CCM-NPD platform One of the key limitations associated with using dissociated cells from neuronal sources directly, without prior purification, is the proliferation of non-neuronal cells. 11,12,34 Since neuronal cells are non-proliferative, non-neuronal cells can outnumber them in medium and long-term cultures, which necessitates a purification process. However, the extended use of harsh antimitotic reagents in inhibitor cocktails, which are commonly used for neuronal purification, can negatively impact neuronal health parameters, such as neurite length.…”

Section: Optimization Of the Ccm-npd Proceduresmentioning

confidence: 99%

“…10 In the studies where non-purified primary neurons were utilized, proliferative glial and fibroblast cells outnumbered primary neurons and competed for nutrients, which gradually reduced neuronal viability. 11,12 Furthermore, these methods are unsuitable for downstream molecular analysis, owing to the contamination from genetic materials and proteins derived from non-neuronal cells. Recently developed techniques have been successfully used for differentiating induced pluripotent stem cells (iPSCs) into desired neuronal cells.…”

Section: Introductionmentioning

confidence: 99%

A fully automated primary neuron purification system using continuous centrifugal microfluidics

et al. 2022

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“…Different kinds of neurons, glial cells, and their fibers in the brain form a complicated network, dominating the normal operation of the body. The neuron is a helpful model in vitro for the research of neurogenesis, synapse formation, and neural circuit formation ( Chen et al, 2013 ; Patel et al, 2019 ). Great emphasis has been placed on avian neurological diseases nowadays, but the lack of suitable infection models in vitro limits the in-depth study of related pathological mechanisms.…”

Section: Introductionmentioning

confidence: 99%