S3 – From evolution and development to regeneration of the central nervous system

This session will delve into the cellular and molecular mechanisms that drive the development and regeneration of the central nervous system (CNS), as well as the impact they had during the evolution of homologous regions of the vertebrate CNS. We will discuss the cellular and molecular mechanisms involved in: conservation of homologies or generation of evolutionary novelties in the hypothalamic region of vertebrates using the prosomeric model to understand the CNS regionalization process; the maintaining and losing of neuro-competence and their relationship to neuro-regenerative capabilities; the formation of topographic ordered connections (mapping); the axon regeneration after injury and its possible implications for planning regenerative medical strategies. These themes not only present a high interest in Neurobiology but also important potential applications in Health Care.

The vertebrate central nervous system, which is built following similar rules in all vertebrates, is the result of a main construction plan (bauplan) that was present at the last common ancestor. A shared bauplan determines a similar regionalization process in the hypothalamic region of south American mammals. However, some differences can be expected in terms of sizes of the main nuclei that make up the hypothalamic region. Following the prosomeric model, the aim of our work was to determine the homologue, but also possible novelties, of the nuclei derived from the hypothalamic region by comparing key mammals from South America. Homologues derivatives are those components that arose from the same part of the bauplan and that may or may be not similar between different species. In that case we analyzed three species with enough evolutive distance to have some potential differences. Adult brains of Didelphis albiventis (Marsupials), Chaetophractus vellosus (Xenarthra) and myotis (microchiropteras) as South American species were selected and compare with mice, rat and gerbil, sectioned and immunohistochemically analyzed with TH (Tyrosine Hydroxylase), PV (Parvalbumin), CR (Calretinin), CB (Calbindin), AVP (Arginine vasopressin), OXT (oxytocin), NF1 (neurophysin 1) and MCH (Melanin concentrating hormone) antibodies. As a result, following the prosomeric model, we compared the topological distribution of some alar and basal plate derivates of the hypothalamic region. We identify homologues components of the paraventricular, supraoptic, arcuate and also for all the specific TH hypothalamic derivatives. Our conclusion was that most of the homologues well know components of the hypothalamic region were present in the species compared. However, there are differences in size and in relation to the radial location of them. Finally, following this analysis some novelties can be proposed. This study could be important for future experimental approaches attempting to understand relevant hypothalamic homeostatic responses in relatively distant South American mammals.

José Luis Ferran Bertone
jlferran@um.es
Departamento de Anatomia Humana y Psicobiologia. Universidad de Murcia, España.
https://portalinvestigacion.um.es/investigadores/333230/detalle

The retinal pigment epithelium (RPE) is a monolayer of cells essential for retina health and physiology. RPE is a plastic tissue that can regenerate neural retina in embryonic amniotes via cell reprogramming. In chicken embryos, RPE reprograms into neural retina after retinectomy and FGF2 stimulation at embryonic day 4 (E4) but not at day 5 (E5), or later. We hypothesized that signaling pathways and intrinsic cell fate control during eye morphogenesis are coupled mechanisms that restrict RPE neurocompetence. To identify transcription factors and signaling pathway candidates for functional perturbation that could promote RPE reprogramming in the late embryonic state, we used single-nucleus multimodal profiling to differentiate chicken RPE from E3-E7. This analysis pointed to 9 up-regulated and 9 down-regulated transcription factor-encoding genes and accompanying changes in motif accessibility that coincided with RPE neurocompetence restriction. Our data suggest that enhanced activity of the Hippo-YAP pathway and transcription factors such as NFIA and NFIB could restrict RPE neurocompetence. Inhibition of the Hippo-YAP pathway significantly increased cell proliferation in E4 and E5 RPE explants in the absence of FGF2, but did not induce retina formation, although it affected the expression of several genes, including EMT regulators and cell cycle-related genes, while suppressing RPE identity genes. In contrast, inhibition of NFIA increased the size of RPE explants, but only at E4. Altogether, our data suggest that the neurocompetence of embryonic RPE cells is jointly regulated by intrinsic and extrinsic cues, with differing effects on RPE cell behavior. Furthermore, these findings indicate that cell proliferation and gene regulatory networks may be responsible for controlling RPE reprogramming and restricting neurocompetence.

Katia Del Rio-Tsonis
delriok@miamioh.edu
Department of Biology, Miami University. Center for Visual Sciences, Miami University, Oxford, OH, USA.
https://www.eyeregenerationlab.com/

Investigating the cellular and molecular mechanisms involved in the development of topographically ordered connections (mapping) of the central nervous system (CNS) constitutes an important issue in neurobiology because these connections are the base of the CNS functions. Furthermore, the regeneration of these connections is the final purpose of any regenerative strategy designed to treat traumatic or degenerative pathologies.
The chicken retinotectal system is the main model to investigate the molecular mechanisms of mapping and the formation of these connections depends of two successive events: 1) the specification of topographic identity of neural progenitors cells (NPC) located in the retina and their targets, which establishes 2) the expression pattern of cellular surface molecules that maintain that topographic identity and direct axon guidance during mapping.
The acquisition of the topographic identity of retinal NPCs is not complete described and this knowledge is of main interest to employ NPC as source of retinal ganglion cells (RGC) which could restitute retino-tectal connections after injury. By culturing neurospheres developed from NPCs obtained from different regions of the retina at different stages of development allowed us to show a critical period for determining topographic identity of NPCs, that FGF2 and insulin are sufficient to obtain RGCs at stages of development when they are not produced and that these RGC are competent to respond to axon guidance cues in a topographic appropriate form. This open the possibility that these RCGs could restitute topographic ordered connections after injury.
On the other hand, the way in which different axon guidance cues may interact and participate in retinotectal mapping is an incompletely understood issue. Thus, we investigated the potential interaction between EphA system and neurotrophins and the intracellular signaling that mediates their effects on axon guidance. We demonstrated that EphA3 and GDNF potentiate nasal RGC axon growth and chemo-attraction by decreasing ephrin-A-mediated EphA4 signaling and increasing FAK. Furthermore, we showed that integrity of lipid rafts is necessary for axon guidance effect mediated by EphA3 and GDNF.
These results highlight about: 1) the combinatorial effects of axon guidance cues during retinotectal mapping; 2) the temporo-spatial pattern and molecular mechanisms of acquisition of topographic identity and cito-differentiation of NPCs to RGCs and; 3) the capabily of RGC to response to axon guidance cues. These data are important in the field of neurobiology and present potential utilities in regenerative medicine.

Gabriel Scicolone
gscicolone@fmed.uba.ar
a. CONICET – Universidad de Buenos Aires, Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis” (IBCN), Ciudad Autónoma de Buenos Aires, Argentina.
b. Universidad de Buenos Aires, Facultad de Medicina, Departamento de Biología Celular, Histología, Embriología y Genética, Ciudad Autónoma de Buenos Aires, Argentina. gscicolone@fmed.uba.ar
https://ibcn.fmed.uba.ar/200_grupos-lab-neurobiologia-scicolone.html

Retinal ganglion cells (RGCs) are the projection neurons of the retina and the first population to be generated in retinal development. In various retinopathies, such as glaucoma, vision loss is a consequence of the progressive dysfunction and regeneration of RGCs and their axons. Atoh7 is an orchestrator of the RGC developmental program and regulates the expression of critical downstream targets, such as POU4F factors. The importance of POU4F factors for RGC development and survival has been established in several studies which showed that the absence of one or more members of this family results in RGC death.

We investigated whether Pou4f2 overexpression in late retinal progenitors (late RPCs) could induce the generation of RGCs beyond their developmental window.

Using a strong ubiquitous promoter to induce Pou4f2 overexpression in neonates through in vivo electroporation, we detected changes in cell distribution in the retina, with increased numbers of electroporated cells in the inner cell layer, where RGCs normally reside. Moreover, we found a high density of projections toward the optic nerve head. Single cell RNA sequencing (scRNA-seq) analysis showed upregulation of multiple RGC-related genes (such as Rbpms, Gap-43, Hs6st3 and Foxp2) following Pou4f2 overexpression. Comparison with previously published scRNA-seq

data from retinal development showed that some cells in the Pou4f2-induced clusters shared similarity with the original RGCs. In addition, gene ontology analysis indicated that axonogenesis and neuronal differentiation were induced after Pou4f2 overexpression. Notably, these RGC-like cells were able to project axons that reached brain targets, such as superior colliculus.

Collectively, these data show that Pou4f2 alone induces a key property of projection neurons that is to project axons up to the brain and endorse its potential as a candidate for reprogramming strategies directed to the generation of new RGCs.

This study was supported by IRRF, CNPq, CAPES and FAPERJ.

Mariana S Silveira
silveira@biof.ufrj.br
Laboratory for Investigation in Neuroregeneration and Development (LINDes), Neurobiology Program, Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brasil.
https://orcid.org/0000-0002-0496-1517