Genetic analyses of thalamocortical axon guidance and subpallial development

Abstract

Normal brain functioning in mammals depends on the establishment of specific, finely-organised neural networks connecting distant regions during development. In particular, sensory stimuli processing is supported by projections from the thalamus to the neocortex. Information from different sensory modalities reaches the thalamus and is here segregated into discrete, specialised nuclei; neurons in each nucleus then send, through topographically-defined axonal connections, elaborated inputs to cognate cortical areas. Our laboratory has previously demonstrated that Semaphorin-6A (Sema6A) exerts key functions in thalamocortical tract formation. During normal brain development, thalamocortical axons (TCAs) extend dorso-laterally through the ventral telencephalon (vTel) to approach the neocortex; axonal navigation in these stages is aided by guiding cell populations, such as the corridor neurons and the guidepost cells of the internal capsule (IC). In Sema6A null mice, however, the caudally-directed dorsal lateral geniculate nucleus (dLGN) axons abnormally extends in the vTel pial surface. How Sema6A influences TCA guidance so specifically has yet to be elucidated. Since interactions between Sema6A, Plexin-A2 (PlxnA2) and Plexin-A4 (PlxnA4) have been implicated in diverse neurodevelopmental processes, we hypothesised that PlxnA2/PlxnA4 might also mediate Sema6A’s dLGN axon guidance functions at subpallial level. In the first part of this study, we therefore focused on characterising thalamocortical tract development in PlxnA2;PlxnA4 double mutants. The analysis of early postnatal mutant brains, performed through axonal marker-specific immunostaining and neuroanatomical tracing techniques, revealed substantial defects in TCA pathfinding which extensively mirrored those observed in Sema6A mutant brains. These findings supported the hypothesised role of Sema6A–PlxnA2-PlxnA4 interactions in TCA guidance. Genetic interaction studies employing Sema6A;PlxnA2 and Sema6A;PlxnA4 mutant mice provided more evidence in favour of this model. In order to understand where and when Sema6A–PlxnA2-PlxnA4 interactions may be required for proper subpallial TCA guidance, we next analysed the spatiotemporal dynamics of Sema6A, PlxnA2 and PlxnA4 expression during early stages of TCA extension in the vTel. We specifically concentrated on characterising expression (i) in thalamic neurons and fibers, and (ii) within intermediate structures that delineate TCA subpallial trajectories, and contain guidepost neural population. mRNA and protein expression were investigated through in situ hybridisation and double immunohistochemistry with known markers for TCAs and specific vTel cell populations. The data we obtained provided novel information on the presence of these guidance molecules in specific mouse forebrain regions from E12.5 to E14.5. Interestingly, we observed that only Sema6A is present in caudally-projecting thalamic neurons, while all proteins are expressed in corridor cells and other structures in the developing basal ganglia. In light of these and earlier findings, we hypothesised that Sema6A–PlxnA2/PlxnA4 interactions could play a role in spatially organising guidepost cell populations that direct TCA growth in the vTel. Thus, we examined whether loss of function of our genes of interest affects the development of corridor and IC guidepost cells. The first neurons were investigated through immunohistochemistry for the marker Islet1 at E12.5–E13.5, while the latter population was analysed (in absence of a specific marker) via neuroanatomical tracing. We observed that in PlxnA2; PlxnA4 and Sema6A mutants some IC guidepost cells are abnormally positioned in correspondence of the ventral route taken by misprojecting TCAs. Furthermore, we reported additional defects in basal forebrain patterning and axon guidance in both mutant mouse lines. Overall, these findings supported our initial speculations on the function of Sema6A, PlxnA2 and PlxnA4 in the spatiotemporal arrangement of TCA guidepost populations in the mammalian vTel. In the final part of this study, we focused on discovering new candidate molecules involved in vTel TCA guidance either as guidance cues, or as regulators of subpallial development and patterning. To this aim, we extracted regional gene expression data from in situ hybridisation experiments collected in the Allen Developmental Mouse Brain Atlas, which represents a set of ~2000 genes analysed at 7 time-points throughout development. Cellular localisation of corresponding protein products per entry was moreover determined using different webtools. With this annotated dataset, we performed an in silico search for genes encoding cell–cell interaction mediators particularly expressed within subpallial structures during early TCA navigation events.