2. Universidad Cardenal Herrera-CEU

The forebrain includes the cerebral cortex, the thalamus, and the striatum and globus pallidus (GP) in the subpallium. The formation of these structures and their interconnections by specific axonal tracts take place in a precise and orchestrated time and spatial-dependent manner during development. However, the knowledge of the molecular and cellular mechanisms that are involved is rather limited. Moreover, while many extracellular cues and specific receptors have been shown to play a role in different aspects of nervous system development, including neuron migration and axon guidance, examples of intracellular signaling effectors involved in these processes are sparse. In the present work, we have shown that the atypical RhoGTPase, Rnd3, is expressed very early during brain development and keeps a dynamic expression in several brain regions including the cortex, the thalamus, and the subpallium. By using a gene-trap allele (Rnd3gt) and immunological techniques, we have shown that Rnd3gt/gt embryos display severe defects in striatal and thalamocortical axonal projections (SAs and TCAs, respectively) and defects in GP formation already at early stages. Surprisingly, the corridor, an important intermediate target for TCAs is still present in these mutants. Mechanistically, a conditional genetic deletion approach revealed that Rnd3 is primarily required for the normal development of Medial Ganglionic Eminence-derived structures, such as the GP, and therefore acts non-cell autonomously in SAs and TCAs. In conclusion, we have demonstrated the important role of Rnd3 as an early regulator of subpallium development in vivo and revealed new insights about SAs and TCAs development.

Neural stem cells directly or indirectly generate all neurons and macroglial cells and guide migrating neurons by using a palisade-like scaffold made of their radial fibers. Here, we describe an unexpected role for the radial fiber scaffold in directing corticospinal and other axons at the junction between the striatum and globus pallidus. The maintenance of this scaffold, and consequently axon pathfinding, is dependent on the expression of an atypical RHO-GTPase, RND3/RHOE, together with its binding partner ARHGAP35/P190A, a RHO GTPase-activating protein, in the radial glia-like neural stem cells within the ventricular zone of the medial ganglionic eminence. This role is independent of RND3 and ARHGAP35 expression in corticospinal neurons, where they regulate dendritic spine formation, axon elongation, and pontine midline crossing in a FEZF2-dependent manner. The prevalence of neural stem cell scaffolds and their expression of RND3 and ARHGAP35 suggests that these observations might be broadly relevant for axon guidance and neural circuit formation.

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.

Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15-day-old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild-types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild-types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild-types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.