The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.
The origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain--especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly "patrolling" for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into "early (amoeboid) microglia": they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier.
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