Control of Orienting Movements and Locomotion by Projection-Defined Subsets of Brainstem V2a Neurons.

Spatial orientation requires the execution of lateralized movements and a change in the animal's heading in response to multiple sensory modalities. While much research has focused on the circuits for sensory integration, chiefly to the midbrain superior colliculus (SC), the downstream cells and circuits that engage adequate motor actions have remained elusive. Furthermore, the mechanisms supporting trajectory changes are still speculative. Here, using transneuronal viral tracings in mice, we show that brainstem V2a neurons, a genetically defined subtype of glutamatergic neurons of the reticular formation, receive putative synaptic inputs from the contralateral SC. This makes them a candidate relay of lateralized orienting commands. We next show that unilateral optogenetic activations of brainstem V2a neurons in vivo evoked ipsilateral orienting-like responses of the head and the nose tip on stationary mice. When animals are walking, similar stimulations impose a transient locomotor arrest followed by a change of trajectory. Third, we reveal that these distinct motor actions are controlled by dedicated V2a subsets each projecting to a specific spinal cord segment, with at least (1) a lumbar-projecting subset whose unilateral activation specifically controls locomotor speed but neither impacts trajectory nor evokes orienting movements, and (2) a cervical-projecting subset dedicated to head orientation, but not to locomotor speed. Activating the latter subset suffices to steer the animals' directional heading, placing the head orientation as the prime driver of locomotor trajectory. V2a neurons and their modular organization may therefore underlie the orchestration of multiple motor actions during multi-faceted orienting behaviors.

Non-thalamic origin of zebrafish sensory nuclei implies convergent evolution of visual pathways in amniotes and teleosts.

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homology is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a thalamic-like sensory structure of teleosts, the preglomerular complex (PG), focusing on the visual projection neurons. Similarly to the tectofugal thalamic nuclei in amniotes, the lateral nucleus of PG receives tectal information and projects to the pallium. However, our cell lineage study in zebrafish reveals that the majority of PG cells are derived from the midbrain, unlike the amniote thalamus. We also demonstrate that the PG projection neurons develop gradually until late juvenile stages. Our data suggest that teleost PG, as a whole, is not homologous to the amniote thalamus. Thus, the thalamocortical-like projections evolved from a non-forebrain cell population, which indicates a surprising degree of variation in the vertebrate sensory systems.

The role of neuro-epithelial-like and radial-glial stem and progenitor cells in development, plasticity, and repair.

Neural stem and progenitor cells (NSPCs) are the primary source of new neurons in the brain and serve critical roles in tissue homeostasis and plasticity throughout life. Within the vertebrate brain, NSPCs are located within distinct neurogenic niches differing in their location, cellular composition, and proliferative behaviour. Heterogeneity in the NSPC population is hypothesized to reflect varying capacities for neurogenesis, plasticity and repair between different neurogenic zones. Since the discovery of adult neurogenesis, studies have predominantly focused on the behaviour and biological significance of adult NSPCs (aNSPCs) in rodents. However, compared to rodents, who show lifelong neurogenesis in only two restricted neurogenic niches, zebrafish exhibit constitutive neurogenesis across multiple stem cell niches that provide new neurons to every major brain division. Accordingly, zebrafish are a powerful model to probe the unique cellular and molecular profiles of NSPCs and investigate how these profiles govern tissue homeostasis and regenerative plasticity within distinct stem cell populations over time. Amongst the NSPC populations residing in the zebrafish central nervous system (CNS), proliferating radial-glia, quiescent radial-glia and neuro-epithelial-like cells comprise the majority. Here, we provide insight into the extent to which these distinct NSPC populations function and mature during development, respond to experience, and contribute to successful CNS regeneration in teleost fish. Together, our review brings to light the dynamic biological roles of these individual NSPC populations and showcases their diverse regenerative modes to achieve vertebrate brain repair later in life.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Lens defects in Astyanax mexicanus Cavefish: evolution of crystallins and a role for alphaA-crystallin.

The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis.

Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

Genome-wide analysis of the POU genes in medaka, focusing on expression in the optic tectum.

The highly conserved POU genes encode homeodomain transcription factors involved in various developmental events, with some, the Brn genes, playing key roles in neurogenesis. We investigated the evolutionary relationships between these genes, by studying the POU gene complement of a model teleost, the medaka (Oryzias latipes). We identified 17 POU genes and carried out a comprehensive in situ hybridization analysis focusing on the optic tectum, a cortical structure of the mesencephalon, in which cell positions and their differentiation states are spatially and temporally correlated. Six POU genes displayed patterned expression in the optic tectum: two genes were expressed in the center of the organ (a zone with differentiated neurons), two in an intermediate zone in which cells exit the cell cycle and two in the peripheral proliferation zone. These results suggest that POU genes may play key roles in both late neurogenesis and in multipotent neural progenitors.

Evidence for neural stem cells in the medaka optic tectum proliferation zones.

Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [1999]: J Comp Neurol 413:385-404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid-binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency-associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio-temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.

Comparison of the expression of medaka (Oryzias latipes) pitx genes with other vertebrates shows high conservation and a case of functional shuffling in the pituitary.

With the availability of an increasing number of whole genome sequences in chordates, exhaustive comparisons of multigene families become feasible. Relationships of orthology/paralogy can not only be inferred from sequence similarity but also by comparing synteny conservation on chromosomes. More accurate scenarios for gene and expression domain gain or loss can now be proposed. Here, we take benefit from the recent release of the medaka (Oryzias latipes) genome to analyse the orthology relationships and expression patterns of the three different sub-families of the pitx homeobox genes belonging to the paired class. They are involved in a wide variety of developmental processes and have pleiotropic expression patterns, especially in the case of the pitx2 sub-family. The emerging picture is a strong conservation of expression domains, suggesting that most functions have been present in the common ancestor of actinopterygians and sarcopterygians. Almost all pitx genes are expressed in anterior placodes in all species studied so far, including medaka. It has previously been shown that in mammals, pitx1 and 2 are expressed in the pituitary. Interestingly we demonstrate here that only pitx3 is expressed in medaka pituitary. It will be interesting to analyze what are the corresponding changes in the regulatory elements of pitx genes.