Continual low-level MEK inhibition ameliorates cardio-facio-cutaneous phenotypes in zebrafish

Abstract

Cardio-facio-cutaneous (CFC) syndrome is caused by germline mutations in KRAS, BRAF and MEK1/2. The highly selective and potent MEK inhibitors that have been developed as anti-cancer agents hold potential as therapeutics for CFC syndrome. We have previously shown that the effects of CFC mutations on zebrafish gastrulation can be prevented by a 1-hour treatment with MEK inhibitors within a specific developmental time-window. However, MEK activity is essential for normal development and PD0325901 treatment outside this treatment window leads to additional developmental defects in MEK-dependent tissues. We now test ten different doses of PD0325901 at six developmental time points and assess the effects on body axis length, heart development and craniofacial structures in zebrafish embryos. Notably, we find that a continuous low-level dose of PD0325901 that has only minor inhibition of MEK activity can prevent the action of both the common CFC BRAF(Q257R) kinase-active allele and the BRAF(G596V) kinase-impaired mutant allele through the first 5 days of development. These results provide a detailed study of the effects of PD0325901 in development and show that, unlike in cancer, which requires robust inhibition of MAPK signalling, a partial reduction in phospho-ERK1/2 activity is sufficient to moderate the developmental effects of BRAF(CFC) mutations.

Fig. 1.

PD0325901 is an effective MEK inhibitor in zebrafish development. (A) Chemical structure of the selective MEK inhibitor PD0325901. (B) Western blot of 10-hpf zebrafish embryos treated with increasing concentrations of PD0325901 from 4 hpf, and immunostained with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. (C) Images of developing zebrafish treated with DMSO, or 0.5 μM or 1.0 μM PD0325901. Zebrafish were imaged under brightfield lighting conditions (top row), or using fluorescence microscopy to visualise GFP expressed from the dusp6 promoter (dusp6-GFP; bottom row). GFP signal was first detected in gastrulating embryos at 50–60% epiboly (white arrowhead). The expression area expanded towards the vegetal pole during epiboly (white arrowhead) and at the tail-bud stage the expression was more diffused with higher expression levels at the tail bud (yellow arrowhead). By 24 hpf, GFP expression was detected in the brain (red arrowhead), along the spinal column and in the tail bud (yellow arrowhead). n=30 embryos per treatment condition.

Fig. 2.

Wild-type embryo sensitivity to PD0325901 treatments. (A) Schematic overview of the design of the PD0325901 treatment regimes. Wild-type embryos were treated with ten increasing concentrations of PD0325901 (0.1–1.0 μM) to identify the most affected tissues at different stages during development. Zebrafish embryos were treated at six different developmental stages (4, 10, 24, 30, 48 and 72 hpf) until 96 hpf. The embryos were monitored daily and were assessed for body axis, cardiac morphology and craniofacial structure phenotypes at 96 hpf. (B) Heat-map of tissue sensitivity to PD0325901 treatment. Each panel shows tissue sensitivity after the addition of increasing concentrations of PD0325901 (0.1–1.0 μM) at a different time in development. Phenotypes were scored for sensitivity to PD0325901, and these data were translated to a colour score in the heat map: 0, no phenotype, blue; +, mild phenotype, yellow; ++, moderate phenotype, orange; +++, severe phenotype, red. PA, pharyngeal arches; MC, Meckel’s cartilage; CH, ceratohyal cartilage; BA, branchial arches. Embryos treated with 0.2 μM PD0325901 from 4 hpf had a normal A-P axis but showed mild malformation of the tail tip (*). The heart valve of embryos treated with 0.1 μM PD0325901 from 48 hpf and with all ten concentrations of PD0325901 from 72 hpf was blocked at 5 dpf (**).

Fig. 3.

Continuous suboptimal PD0325901 treatment prevents zebrafish BRAF^CFC phenotypes. (A) Wild-type embryos were injected with human BRAF^WT, the kinase-activating and most common CFC allele BRAF^Q257R, or the kinase-inactivating BRAF^G596V, and imaged at the indicated developmental time post-fertilisation. (B) Images of untreated control zebrafish (top panel) and a zebrafish expressing the kinase-inactivating BRAF^G596V allele under the melanocyte-specific mitfa promoter (bottom panel). Insert image depicts a high-magnification image of ectopic melanisation and nevi formation. (C) Continuous treatment of BRAF^WT- and BRAF^CFC-injected embryos with a suboptimal dose (0.2 μM) of PD0325901 from 4 hpf through 4 dpf. (D) Western blot of 10-hpf wild-type [uninjected (Un)] or BRAF^CFC embryos treated with 0.2 μM of PD0325901 from 4 hpf, and immunostained with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies.