Zika virus (ZIKV) infection during pregnancy is linked to microcephaly, which is attributed to infection of developing brain structures. ZIKV infects neural progenitor cells in vitro, though its effects on other developmentally relevant stem cell populations, including cranial neural crest cells (CNCCs), have not been assessed. CNCCs give rise to most cranial bones and exert paracrine effects on the developing brain. Here, we report that CNCCs are productively infected by ZIKV, but not by the related dengue virus. ZIKV-infected CNCCs undergo limited apoptosis but secrete cytokines that promote death and drive aberrant differentiation of neural progenitor cultures. Addition of two such cytokines, LIF or VEGF, at levels comparable to those secreted by ZIKV-infected CNCCs is sufficient to recapitulate premature neuronal differentiation and apoptotic death of neural progenitors. Thus, our results suggest that CNCC infection by ZIKV may contribute to associated embryopathies through signaling crosstalk between developing face and brain structures.

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Despite productive infection of CNCCs with ZIKV, relatively little apoptotic death is seen following ZIKV infection. We observe a minor increase in apoptotic death at 24 hr after ZIKV exposure (4.3% compared to 3.6% at baseline), but not for DENV (2.9% compared to 3.6%) ( Figure 1 F). This effect is slightly more pronounced at 72 hr after exposure, with 8.8% of the ZIKV virus-infected cells undergoing apoptotic death, compared to 4% apoptosis with mock infection and 3.9% with DENV ( Figure 1 G). A lower MOI leads to a slight decrease in levels of apoptosis ( Figure S1 E). Taken together, these observations indicate that CNCCs are highly susceptible to ZIKV infection and support viral replication while only experiencing modest apoptotic death. Interestingly, this susceptibility is specific, as CNCCs are resistant to infection by a related flavivirus, DENV.

+DAPI+ protrusions migrating away from the sphere bolus (+ axonal projections ( In light of the importance of CNCCs in establishing the signaling milieu in the developing brain, we next examined whether the paracrine effects of ZIKV-infected CNCCs could have an effect on developing NPCs. To this end, CNCC monolayers were seeded on transwell membranes and infected with ZIKV (or mock infected) for 24 hr before co-culturing with neurospheres separated by the transwell membrane. After 3 days, neurospheres were fixed and visualized by confocal immunofluorescence imaging. Migratory projections were visualized as Tuj1DAPIprotrusions migrating away from the sphere bolus ( Figure 1 Hi), increased neuronal growth as Tuj1axonal projections ( Figure 1 Hii), and apoptosis by cleaved caspase-3 staining ( Figure 1 H, green). Neurospheres co-incubated in with ZIKV-infected, but not uninfected, CNCCs across a transwell membrane showed striking changes in morphology, including presence of migratory or neuronal projections ( Figure 1 H; quantified in Figure 1 I). Notably, the pronounced morphological differences seen after co-incubation with infected CNCCs are not seen following direct infection of neurospheres with ZIKV ( Figure S1 F). In addition, apoptosis is significantly increased in neurospheres co-incubated with infected CNCCs, compared to neurospheres cultured alone ( Figures 1 H and 1J).

Our observation that ZIKV causes productive infection in CNCCs is significant on several levels. First, size and development of the skull are affected in ZIKV-associated microcephaly. While this may be an indirect consequence of defective brain growth, in light of our results, direct impact of ZIKV infection on cranial mesenchyme should be considered. Second, we showed that ZIKV can replicate in CNCCs and that the majority of ZIKV-infected CNCCs do not seem to undergo apoptosis, and thus these cells (and potentially their derivatives) could act as a reservoir for ZIKV reproduction in the vicinity of the developing brain, allowing for continued secretion of infectious virus. Since no infection or viral replication by DENV is seen, the resistance of CNCCs to DENV may provide one explanation for the lack of significant adverse fetal outcomes following maternal DENV infection. Finally, because the development of CNCCs and their derivatives is closely intertwined with the formation of the CNS, our data uncover a potential mechanism by which ZIKV infection may drive embryonic pathogenesis in the brain itself. Considerable increases in secretion of LIF, VEGF, IL-6, and other molecules by ZIKV-infected CNCCs could have both autocrine effects (in turn affecting formation of cranial bone and cartilage) as well as paracrine effects on the developing CNS. Indeed, we provide evidence that addition of either LIF or VEGF alone at concentrations elicited by ZIKV infection influences survival and differentiation of NPCs. Thus, our work brings attention to previously unappreciated non-cell-autonomous mechanisms that may underlie ZIKV-associated microcephaly.