Origins and unconventional behavior of neutrophils in developing zebrafish

Abstract

The first leukocytes that arise in the development of vertebrate embryos are the primitive macrophages, which differentiate in the yolk sac and then quickly invade embryonic tissues. These macrophages have been considered to constitute a separate lineage, giving rise to no other cell type. Using an in vivo photoactivatable cell tracer in the transparent zebrafish (Danio rerio) embryo, we demonstrated that this lineage also gave rise to an equal or higher number of neutrophilic granulocytes. We were surprised to find that the differentiation of these primitive neutrophils occurs only after primitive myeloid progenitors have dispersed in the tissues. By 2 days after fertilization, these neutrophils have become the major leukocyte type found wandering in the epidermis and mesenchyme. Like the primitive macrophages, all primitive and larval neutrophils express PU.1 and L-plastin and they are highly attracted to local infections, yet only a small fraction of them phagocytose microbes, and to a much lesser extent per cell than the macrophages. They are also attracted to variously stressed or malformed tissues, suggesting a wider role than antimicrobial defense.

NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation

“…1b), and no neutrophils in the CHT (Fig. 1d), which normally produces numerous neutrophils by this stage 9 (Fig. 1a,c).…”

“…VE-DIC and fluorescence microscopy were performed as described previously 6,9 through the 60x/1.00 NA water-immersion objective of a Nikon 90i microscope. Time-lapse recording of VE-DIC images was performed using BTVpro software (Bensoftware, London).…”

“…in 4% methanol-free formaldehyde overnight then kept in methanol at À 20°C for at least 2 h. TUNEL staining was performed using ApopTag Red or Fluorescein In Situ Apoptosis Detection Kit (Chemicon) combined with peroxidase (POD) coupled anti-DIG antibody (Roche #11207733910, 1:200) followed by tyramide-based amplification 9 . To visualize the transgenic reporter proteins, rabbit anti-GFP (MBL #598, 1:300) or rabbit antiDsRed/mCherry (Clontech #63296, 1:300) antibodies were co-incubated with the POD-coupled anti-DIG antibody, then revealed by anti-rabbit-AlexaFluor 488 (Life Technologies; #A11070, 1:200) and anti-rabbit-Cy3 (Jackson Lab; #711166152, 1:300) coupled antibodies, except for the [TCF:nls-mCherry] larvae, which were subjected to tyramide-based detection of POD-coupled anti-rabbit antibodies (Life Technologies #G21234, 1:300).…”

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation

“…O-dianisidine was performed as previously described[24]. Embryos treated with 1-phenyl 2-thiourea (PTU) to prevent pigmentation were stained with Sudan Black as previously described[25]. TUNEL staining was performed according to manufacturer's instructions using Millipore's ApopTag® Plus Peroxidase In Situ Apoptosis Kit.…”

“…TUNEL staining was performed according to manufacturer's instructions using Millipore's ApopTag® Plus Peroxidase In Situ Apoptosis Kit. Globin antibody staining was performed as described[25] using a zebrafish antibody from Anaspec (Anti-Hbbe, Catalog Number 55608).…”