Exploring the Expression and Function of cTyro3, a Candidate Zika Virus Receptor, in the Embryonic Chicken Brain and Inner Ear

Abstract

The transmembrane protein Axl was proposed as an entry receptor for Zika virus (ZIKV) infection in vitro, but conflicting results from in vivo studies have made it difficult to establish Axl as a physiologically relevant ZIKV receptor. Both the functional redundancy of receptors and the experimental model used can lead to variable results. Therefore, it can be informative to explore alternative animal models to analyze ZIKV receptor candidates as an aid in discovering antivirals. This study used chicken embryos to examine the role of chicken Tyro3 (cTyro3), the equivalent of human Axl. Results show that endogenous cTyro3 mRNA expression overlaps with previously described hot spots of ZIKV infectivity in the brain and inner ear. We asked if ectopic expression or knockdown of cTyro3 influenced ZIKV infection in embryos. Tol2 vectors or replication-competent avian retroviruses were used in ovo to introduce full-length or truncated (presumed dominant-negative) cTyro3, respectively, into the neural tube on embryonic day two (E2). ZIKV was delivered to the brain 24 h later. cTyro3 manipulations did not alter ZIKV infection or cell death in the E5/E6 brain. Moreover, delivery of truncated cTyro3 variants to the E3 otocyst had no effect on inner ear formation on E6 or E10.

Keywords: Axl; Tyro3; Zika; basilar papilla; neural tube; otocyst.

Figure 1

Endogenous cTyro3 mRNA expression pattern in the chick embryonic brain overlaps with known hotspots of ZIKV infection. (A) Sections from embryos sampled daily from E2 to E5 stained with cTyro3 mRNA probe using RNAscope in situ hybridization (representative of n = 2–6 per day). cTyro3 expression is strong in regions known to be readily infected with ZIKV, including the floor plate of the hindbrain (closed arrowheads). cTyro3 mRNA expression of a ZIKV-infected E5 embryonic chick brain in (B) the diencephalon and (C) the midbrain shows regions with overlapping ZIKV infection in alternate sections (B’) and (C’) respectively (closed arrowheads). Surrounding regions that did not show overlap are indicated with open arrowheads. Mesenchyme surrounding the brain, previously shown to be permissive for ZIKV, strongly expresses cTyro3 and, in the ZIKV-infected embryo of panel (C’), is positive for dsRNA. Abbreviations: A, anterior; DE, diencephalon; FB, forebrain; HB, hindbrain; MB-FP, midbrain floor plate; MB-RP, midbrain roof plate; mes, mesenchyme; P, posterior.

Figure 2

Endogenous cTyro3 mRNA expression levels in the embryonic chick inner ear. (A–H) cTyro3 mRNA expression in the inner ear was visualized at E2–E6, E7.5, E8 and E10–11 (n = 1 to 6 per specified day). Expression level is low at (A) E2 when the otic cup invaginates (open arrowheads) but increases with age (B–D) as the inner ear sensory organs begin to form. Positive staining is particularly robust in the anlage of the basilar papilla, the auditory sensory organ in birds (boxed regions at E6 and E7.5–8). (E–H) The sensory region of the basilar papilla (BP) has exceptionally high cTyro3 mRNA expression from E6-E10. (H) The supporting cell layer of the BP has somewhat higher cTyro3 mRNA expression than the hair cell layer at E10. Abbreviations: BP, basilar papilla; E, embryonic day; Hm, non-sensory homogene cells; HCL, hair cell layer; SCL, supporting cell layer.

Figure 3

cTyro3 constructs successfully express expected proteins. Western blot analysis of transfected lysates and supernatants in DF-1 cells to confirm protein expression of (A) full-length cTyro3 and (B) truncated HA-tagged cTyro3 mutants. (A) Full-length cTyro3 has intact kinase activity as shown by PY20 antibody signal that detects phosphorylated tyrosine residues only in cells transfected with the cTyro3 plasmid. GFP is present in both the cTyro3-GFP and control GFP transfected lysates. (C) Transfected cells were fixed and immunostained for GFP or HA.11 antibodies to confirm protein expression in cells. For these and other images with GFP labeling, the endogenous GFP fluorescence signals were enhanced by immunostaining for the protein using a secondary antibody with green fluorescence. Scale bar = 100 µm. Abbreviations: kDa, kiloDaltons.

Figure 4

Ectopic cTyro3 overexpression or truncation does not correlate with the locations of ZIKV infection. (A) Timeline of the experiment to test the effect of cTyro3 overexpression on ZIKV infection in the embryonic brain. (B) Experimental methodology for expressing plasmids encoding bicistronic mRNA to encode full-length cTyro3 and GFP proteins, and designed for stable integration and expression in chick embryos using a transposase expression plasmid. Both plasmids are electroporated to the right side of the neural tube at E2 (black triangle, 2 dpi) followed by ZIKV injection into the midbrain ventricle 24 h later (blue triangle, Z). (C–F) Analysis of ZIKV infection in the presence of ectopic cTyro3 from 18 embryos. (C) Co-localization of GFP protein and cTyro3 mRNA after electroporation was confirmed using immunohistochemistry and RNAscope in situ hybridization, respectively, on alternate sections at E5. Note how high cTyro3 over-expression appears in the dorsal midbrain in comparison to endogenous expression levels. (D–F) A dsRNA antibody was used to localize ZIKV infection in the red channel (shown in grey scale). ZIKV infection did not consistently overlap with ectopic cTyro3 transfection (visualized by GFP in the green channel) as overlap could be seen for both (D) the control (n = 1/5 embryos) as well as (E) the experimental samples (n = 3 embryos). (F) Regions of the midbrain that did not have any ectopic cTyro3 also showed ZIKV infection (open arrowheads) while cTyro3-transfected cells (green) nearby had no infection (n = 12 embryos). Scale bar = 100 µm. (G) Timeline of experiment to test the effect of cTyro3 dominant-negative mutants on ZIKV infection. RCAS viral vectors encoding the truncated cTyro3 mutants were injected into the E2 neural tube (black triangle, 2 dpi), followed by ZIKV injection one day later (blue triangle, Z). (H–K) Known hotspots of ZIKV infection were visualized for reduction in infection compared to RCAS control embryos. ZIKV infection levels, already relatively low in these experiments, did not appear to be blocked in areas transfected with cTyro3 mutant constructs. Open arrowheads show HA.11-positive regions with no corresponding ZIKV infection while closed arrowheads show regions with ZIKV infection despite expressing a dominant-negative receptor. Images are representative of trends seen for n = 8 embryos screened for the cTyro3_TM_Ex mutant and n = 6 embryos screened for the cTyro3_Ex mutant. Scale bar = 50 µm. Abbreviations: DE, diencephalon; E, embryonic day; FB, forebrain; HB, hindbrain; MB, midbrain; MB-RP, midbrain roof plate.

Figure 5

Expression of cTyro3 dominant negatives does not affect cell death and hair cell expression in the inner ear. (A) Timeline of experiment. ZIKV was injected on E3 (green triangle) with embryos processed on E3 or E10 (black triangles) (B–E) Infection of the right inner ears with RCAS(A) is indicated by immunostaining of viral gag protein with 3C2. Infection does not change immunostaining for activated Caspase 3 in (B) the fusion plate (FP) of the lateral canal (open arrowheads) or the (C) ventromedial hotspot of programmed cell death (VM-PCD) (closed arrowheads), when compared with the same regions in the uninfected left ear. (D,E) Expression of the hair cell marker HCS-1 is unchanged on the infected right side compared to the uninfected left side. In (E), the tectorial membrane is non-specifically labelled with HA.11 (green) antibody in both ears. Scale bar = 100 µm. Abbreviations: BP, basilar papilla; dpi, days post-infection; FP, canal fusion plate; HCL, hair cell layer; SAG, statoacoustic ganglion; SCL, supporting cell layer; Tm, tectorial membrane; VM-PCD, ventromedial hotspot of programmed cell death.