Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to oncogene activation in melanocytes

Abstract

A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes (NRasQ61K, HRasG12V and KRasG12V) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma in vivo, revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma.

Inflammatory Responses during Tumour Initiation: From Zebrafish Transgenic Models of Cancer to Evidence from Mouse and Man

“…Subsequent zebrafish studies have shown a similarly rapid recruitment of neutrophils and/or macrophages to the skin, liver and brain in response to transgenic expression of numerous oncogenes, e.g. HRAS G12V , KRAS G12V , NRAS Q61K , Src, Xmrk, Myc and Akt [6,7,[39][40][41]60,68,104,[140][141][142][143]. Live imaging has provided insight into the dynamics of the intrinsic inflammatory response following tumour initiation within these models.…”

“…Transcriptomic analysis of PNC-associated neutrophils in an NRAS Q61K melanoma model, showed upregulation of genes that correspond with growth-and invasion-promoting properties, e.g. FGF-1 and -6, cathepsin-H and galectin1 -and -3 [60].…”

“…In addition to promoting PNC proliferation, zebrafish cancer models have also provided evidence that genes involved in angiogenesis and invasion are expressed at the pre-neoplastic stage by both PNCs and inflammatory cells [60,104,140,144]. This is of interest since these processes are traditionally associated with later stages of cancer but are likewise difficult to capture at early stages in mammalian models [2].…”

Biotagging, an in vivo biotinylation approach for cell-type specific subcellular profiling in zebrafish

“…It has been previously reported that in vivo biotinylation in skeletal muscle cells requires biotin supplementation in the zebrafish embryo media [26]. However, biotin supplementation has not been necessary in a number of tissues in which biotagging has been performed [31,44,45]. The differences in endogenous levels of biotin may be due to variations in maternal diet.…”

Re-purposing Ac/Ds transgenic system for CRISPR/dCas9 modulation of enhancers and non-coding RNAs in zebrafish

“…Buoyed by this important finding, Ac/Ds elements were employed to facilitate the integration of a reporter construct into the zebrafish genome with high efficiency, leading to remarkable germline transmission rates (Emelyanov et al, 2006). This approach was the integration method of choice used to generate transgenic zebrafish lines for the chemical-inducible LexPR transactivation system (Emelyanov and Parinov, 2008, Kenyon et al, 2018), as well as for a systematic mutagenesis gene-trapping screen (Quach et al, 2015). While these studies demonstrated the utility of Ac/Ds as an efficient method for propagation of transgenes through the germline, they overlooked its strong potential for transient expression of DNA elements in F 0 embryos, which is a current limitation of the zebrafish model.…”