Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants

Area of Science:

• Developmental biology
• Cellular mechanics
• Zebrafish model system

Background:

• Zebrafish embryos are ideal for studying organogenesis due to optical clarity and rapid development.
• Extended imaging is hindered by embryonic axis changes, movement artifacts, and yolk obstruction.
• The yolk cell impedes optical access to critical embryonic tissues.

Purpose of the Study:

• To develop a method for removing the zebrafish yolk cell to improve imaging.
• To enable detailed cellular and mechanical studies of organogenesis.
• To facilitate high-resolution imaging of embryonic tissues.

Main Methods:

• Injection of AMP-PNP (adenosine triphosphate analog) to paralyze the yolk cell.
• Mechanical removal of the yolk cell using tungsten needles.
• Immobilization of deyolked embryos or explants on coverslips using a plasma clot for advanced microscopy.

Main Results:

• Deyolked embryos and explants allow for novel imaging orientations (ventral, lateral, cross-sectional) with high numerical aperture objectives.
• Isolated head rudiments maintain normal morphogenesis and gene expression in organotypic culture for over 24 hours.
• The technique is compatible with green fluorescent protein transgenic animals.

Conclusions:

• Yolk cell removal significantly enhances optical access for studying zebrafish organogenesis.
• This method provides a robust platform for investigating cellular mechanics in deyolked embryos and explants.
• The technique supports long-term organotypic culture and high-resolution imaging of embryonic development.