Congenital eye malformations associated with extensive periocular neural crest apoptosis after influenza B virus infection during early embryogenesis

Abstract

Purpose: Congenital eye malformations are a leading cause of blindness in children. Influenza virus infections prevail worldwide and have been implicated in congenital defects. Infections acquired during gestation may disrupt eye morphogenesis. We investigated the effects of influenza B virus infection on eye malformations during early embryogenesis.

Methods: Chick embryos were exposed to influenza B virus at Hamburger-Hamilton stage 9. Maternal infection was conducted by exposing pregnant ICR mice to influenza B virus at the embryonic gestation stage E 5.0. After infection, virus RNA distribution was detected by in situ hybridization at various developmental stages. The distribution of periocular neural crest cells and the extent of apoptosis were examined by immunohistochemical staining, in correlation with eye malformations.

Results: Microphthalmos and anophthalmos, together with neural tube defects, were found in the chick and mouse embryos following the infections. The viral RNA was detected in the head neuroepithelium, optic vesicle, periocular mesenchyme, and the forming ventricles of the developing brain. Immunohistochemical staining revealed aberrant neural crest distribution and extensive apoptosis in the head surface ectoderm, periocular mesenchyme, and optic vesicle in the chick embryos. Furthermore, transplacental infection was confirmed by the presence of viral RNA in the mouse fetuses, with eye and neural tube defects similar to those found in the chick embryos after experimental infections.

Conclusions: Influenza B virus may act as a teratogen to cause aberrant periocular neural crest cell contribution to eye development and extensive apoptosis, resulting in congenital eye malformations.

Figure 1

Eye malformation and neural tube defect after influenza B virus infection in the chick embryo model. A, E, G, I, and K were from sham-infected control embryos. B, C, and D were from an infected embryo. F, H, J, and L were from another infected embryo. A-D were processed through in situ hybridization with a RNA probe containing influenza B virus hemagglutinin segment. The presence of viral RNA was indicated by blue or purple signals. E-H were processed through in situ apoptosis assay on transverse tissue sections, with apoptotic cells indicated by green fluorescence signals. J-L were also processed through in situ apoptosis assay on transverse tissue sections, but without adding terminal deoxynucleotidyl transferase. A: A sham-infected control embryo. B: Dorsal view of an infected embryo exhibited abnormal optic vesicle development (indicated by arrows and dotted line), as compared to the control embryo showing normal optic vesicle development in A (indicated by arrows and dotted line). The viral RNA was detected in the head neuroepithelium, optic vesicle, periocular mesenchyme, and in the heart. C: Ventral view of the same embryo in B, showing evident presence of viral RNA in the head neuroepithelium. D: Coronal section of the embryo in B, showing the presence of viral RNA in the neural tube (indicated by arrowhead) and the forming ventricles of the developing brain enclosed by twisted neuroepithelium (indicated by arrows). E and G: Midbrain and hindbrain sections of a sham-infected control embryo showing a few apoptotic cells normally observed during early chick embryogenesis. F and H: Extensive apoptotic cells in the midbrain and hindbrain sections of a virus-infected embryo. I and K: Negative controls for TUNEL assay on sham-infected embryos corresponding to E and G and showing no background signals. J and L: Negative controls for TUNEL assay on virus-infected embryos corresponding to F and H and showing no background signals. Abbreviations: fb, forebrain; hb, hindbrain; ht, heart; mb, midbrain; mes, mesenchyme; nep, neuroepithelium; opv, optic vesicle. Scale bar: 50 µm.

Figure 2

Teratogenic effects of influenza B virus infection in the chick embryo model. The images in A-J are from the same embryo, and those in K-R are from another embryo. A-J: A virus-infected embryo at Hamburger-Hamilton stage 15 showing gross malformations (A-D), localization of viral RNA on whole mount preparation (E and F) and on tissue sections (G-J). K-R: Distribution of HNK-1 positive neural crest cells in a virus-infected embryo at Hamburger-Hamilton stage 13 showing neural crest cell population in the dorsal view (K), ventral view (L), lateral views (M and N), and in tissue sections (O-R). S and U: Forebrain and hindbrain sections of a sham-infected control embryo at Hamburger-Hamilton stage 15 showing few apoptotic cells. T and V: Extensive and unilateral distribution of apoptotic cells in the forebrain and hindbrain sections of a virus-infected embryo at Hamburger-Hamilton stage 15. W: A negative control for TUNEL assay on sham-infected embryo corresponding to S and showing no background signals. X: A negative control for TUNEL assay on virus-infected embryo corresponding to T and showing no background signals. A and B: Lateral view of asymmetric eye development (indicated by white arrowhead and white arrow) and twisted neural tube in the same embryo. C and D: Magnified head region showing asymmetric eye development. E (on focus) and F (out of focus): Asymmetric eye development and unilateral distribution of viral RNA as shown by the blue signals on the same focus plane. G-J: Areas of transverse sections as indicated in E, showing evident unilateral distribution of viral RNA in the head mesenchyme (indicated by arrow). Note that viral RNA was concentrated on the side of eye malformation (indicated by arrowheads in H). K-N: Asymmetric distribution of neural crest cells in a virus-infected embryo. The normal eye (arrow-indicated) was surrounded by more neural crest cells than the malformed eye (arrowhead indicated). O-R: more evident asymmetric neural crest cell distribution, as shown in blue signals, was seen on sections of the areas indicated in M. The asterisk in O indicates unilateral shortage of neural crest cells as compared to the other side. T and V: More apoptotic cells were detected in the surface ectoderm, neuroepithelium, and mesenchyme as compared to the sham-infected controls (S and U). Abbreviations: ba, branchial arch; fb, forebrain; hb, hindbrain; ht, heart; mb, midbrain; mes, mesenchyme; ov, otic vesicle. Scale bars: F and N, 200 μm; J and R: 100 μm; V and X, 50 μm.

Figure 3

Teratogenic effects of influenza B virus infection in the mouse embryo model. A and F are from sham-infected control embryos. B-E and G-K are from virus-infected embryos. A-G are wholemount embryo preparations. J-K are embryo tissue sections. A: A mouse embryo showing normal development at E9.5 after sham infection at E5.0. B: Reduction of embryo size in 3 virus-infected embryos as compared to the sham-infected control in A at the same gestation stage. C: The blue signals indicate the localization of viral RNA in neuroepithelium and as indicated by arrowheads, in an infected mouse embryo. D: An example of abnormal optic vesicle (indicated with arrowhead), as compared to the normal optic vesicle in A (indicated with arrow) at the same gestation stage. E and G: Twisted head neuroepithelium and trunk neural tube (indicated by arrowheads) as compared to the normal neural tube in F. H: Viral RNA distribution in embryonic endoderm (indicated by arrowheads). I: Viral RNA distribution in the amniotic membrane (indicated by arrows) and in placenta (indicated by arrowheads). J: Viral RNA distribution in trunk surface ectoderm (indicated by arrowheads) and in the neural tube (indicated by an arrowhead). K: A negative control for the in situ hybridization procedures showing no background signals. Gestation stages of embryos: A-G, E9.5; H, E6.0; I and J, E6.5. Abbreviations: ba, branchial arch; ee, embryonic endoderm; etc, ectoplacental cone; fb, forebrain; hb, hindbrain; ht, heart; nt, neural tube; mes, mesenchyme; opv, optic vesicle; ov, otic vesicle; pl, placenta. Scale bars: A, B, C, and E, 500 μm; F and G, 50 μm; H-K, 100 μm.