Transgenic zebrafish reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages

Abstract

The immune response of a host to an invading pathogen is dependent on the capacity of its immune cell compartment to recognize highly conserved pathogen components using an ancient class of pattern recognition receptors known as Toll-like receptors (TLRs). Initiation of TLR-mediated signaling results in the induction of proinflammatory cytokines that help govern the scale and duration of any ensuing response. Specificity for TLR signaling is, in part, a result of the differential recruitment of intracellular adaptor molecules. Of these, MyD88 is required for the majority of TLR signaling. Zebrafish have been shown to possess TLRs and adaptor molecules throughout early development, including MyD88, strongly suggesting conservation of this ancient defense mechanism. However, information about which embryonic cells/tissues possess this conserved signaling potential is lacking. To help define which embryonic cells, in particular, those of the innate immune system, have the potential for MyD88-dependent, TLR-mediated signaling, we generated transgenic reporter lines using regulatory elements of the myd88 gene to drive the fluorescent reporters enhanced GFP and Discosoma red fluorescent protein 2 within live zebrafish. These lines possess fluorescently marked cells/tissues consistent with endogenous myd88 expression, including a subset of myeloid leukocytes. These innate immune cells were confirmed to express other TLR adaptors including Mal, trif, and Sarm. Live wound-healing and infection assays validated the potential of these myd88-expressing leukocytes to participate in immune responses. These lines will provide a valuable resource for further resolving the contribution of MyD88 to early vertebrate immunity.

HNF1β Is Essential for Nephron Segmentation during Nephrogenesis

“…The cadherin-17 (cdh17) gene was chosen because it is highly expressed in all tubule segments. 11 Although a 4.6 kb cdh17 promoter was initially identified, 12 we subsequently found that 1.2 kb of genomic sequence upstream of the start codon is equally sufficient ( Figure 1A). The spatiotemporal expression of this shorter promoter recapitulates the endogenous cdh17 gene expression pattern with eGFP fluorescence/transcripts being detected in all tubule segments from the 8-somite stage to 3 days postfertilization (dpf) (Supplemental Figure 1, A-C).…”

Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88

“…The zebrafish genome has duplicated tlr genes but only a single copy of myd88 (21,22). We and others have shown that Myd88 functions in zebrafish to modulate innate immune responses to microbes and microbial-associated molecular patterns (MAMPs), such as LPS (23)(24)(25). Notably, LPS sensing in zebrafish differs mechanistically from that in mammals and does not involve a Tlr4-MD2 complex (26).…”

Regulation of immunity and disease resistance by commensal microbes and chromatin modifications during zebrafish development

“…Thus, they are able to sense the same ligands (15)(16)(17), use similar adaptor molecules for signaling (15), and activate the transcription factor NF-κB (13,15,18). In addition, it has been shown that in zebrafish MyD88 modulates innate immune responses to microbes (19)(20)(21)(22).…”