Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Neurospheres obtained from the ciliary margin of the chicken eye possess positional values and retinal ganglion cells differentiated from them respond to EphA/ephrin-A system.

In the Central Nervous System (CNS) there are some niches of undifferentiated, neural progenitor/stem cells that produce active neurogenesis originating functionally integrated neurons. In the chicken eye, there is a neurogenic niche in the ciliary margin (CM) which has the ability to originate all the cell types of the neural retina. During retinal development, cells acquire positional values along the radial and tangential axes. These positional values are the necessary base for the formation of neural circuits. In this work, we have analyzed whether neural progenitor cells (NPCs) of CM have positional values regarding the radial and tangential axes, and if they have the potential to differentiate into retinal ganglion cells (RGCs) in vitro. Furthermore, we analyzed whether these RGCs preserve positional values along the tangential axis and respond to the Eph/ephrin axon guidance system. In order to answer these questions, we cultured NPCs obtained from the CM favoring the formation of neurospheres. Our results showed that the expanding neurospheres are polarized structures in which their cells have specific positional values along their radial axis, recapitulating the apical-basal polarity of the CM and the neuroepithelium. We also showed that NPCs obtained from CM possess positional values along the nasal-temporal retinal axis. When the neurospheres were submitted to differentiation conditions, we observed that NPCs can differentiate into RGCs. These RGCs present long axons that express different members of the Eph/ephrin system and they are competent to respond to this axon guidance cue system, recapitulating the axonal behavior during retinotectal neural map development. All these findings contribute to understand the cellular and molecular mechanisms involved in CNS development and regeneration.

Molecular complexity of visual mapping: a challenge for regenerating therapy.

Investigating the cellular and molecular mechanisms involved in the development of topographically ordered connections in the central nervous system constitutes an important issue in neurobiology because these connections are the base of the central nervous system normal function. The dominant model to study the development of topographic maps is the projection from the retinal ganglion cells to the optic tectum/colliculus. The expression pattern of Eph/ephrin system in opposing gradients both in the retina and the tectum, labels the local addresses on the target and gives specific sensitivities to growth cones according to their topographic origin in the retina. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. However, several lines of evidence have shown that the mapping can adjust to experimentally modified targets with flexibility, demonstrating the robustness of the guidance process. Here we discuss the complex ways the Ephs and ephrins interact allowing to understand how the retinotectal mapping is a precise but also a flexible process.

Regulation of axonal EphA4 forward signaling is involved in the effect of EphA3 on chicken retinal ganglion cell axon growth during retinotectal mapping.

The Eph and ephrins are involved in the genesis of topographic ordered connections at the visual system. Previously we demonstrated that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) toward the caudal tectum preventing them from branching in the rostral tectum. Now we investigated whether tectal EphA3 plays this role by modulating the axonal EphA4 forward signaling or throughout axonal ephrin-As reverse signaling. For this purpose we used cultures of nasal retinal explants and dissociated retinal neurons from chicken embryos. We treated them with clustered EphA3-Fc, Fc (control), PI-PLC (sheds ephrin-As) or KYL (inhibits ephrin-As-mediated EphA4 activation). We achieved in vitro and in vivo electroporations of chicken embryo retinas with wild type EphA4, Ki-EphA4 (kinase inactive dominant negative EphA4) or EGFP in pMES expression vector. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrin-As system. Our results showed that: 1) shedding of ephrin-As and the inhibition of ephrin-A-mediated EphA4 activity increase axon length and decrease axonal interstitial filopodia density of nasal RGCs; and 2) a dominant negative form of EphA4 increases axon growth in vitro and induces nasal RGC axons to grow passing throughout their target area in the caudal tectum meanwhile overexpression of EphA4 produces the opposite effects. All together, these results demonstrate that ephrin-A-mediated EphA4 forward signaling decreases the level of axon growth and increases the density of axonal interstitial filopodia of nasal RGCs. Besides, our results showed that: 3) EphA3 ectodomain increases axon growth and decreases the density of axonal interstitial filopodia and branching in vitro and in vivo and 4) EphA3 ectodomain diminishes the ephrin-A2/EphA4 colocalization, and the EphA4 and ephexin1 phosphorylation. All together, these results show that the EphA3 ectodomain produces the opposite effects than the EphA4 forward signaling, by decreasing this signaling pathway throughout competing with EphA4 for ephrin-As binding. Furthermore, it is proposed that tectal EphA3 participates in the establishment of retinotectal mapping throughout this mechanism and that EphAs can regulate axon growth and branching by modulating other EphA receptors forward signaling.