Final Report Summary - NEUROMIGRATION (Novel Molecular Mechanisms of Neuron Migration in the Developing Cortex and their Contribution to Related Diseases)
Understanding the nervous system function is probably the most challenging goal for the XXIst century in biomedical research. Most of the nervous system connectivity is established during development as a result of a delicate coordination of neuron migration, axon guidance and synapse formation. These processes are ultimately regulated by molecules (for instance, the axon guidance cues), that interact with specific receptors at the surface of the neuron. However, the signaling mechanisms involved are not fully characterized. The objective of this project was to assess the role of a family of leucine-rich transmembrane proteins (LRTPs), the so-called fibronectin and leucine-rich transmembrane proteins (FLRT1-FLRT3) during nervous system development in vivo and of the intracellular mechanisms involved. We also wished to expand these observations to other LRTPs in the developing brain both in mice and humans.
Traditionally, the effects of the axon guidance molecules and the intracellular mechanisms involved have been studied individually. However, it has been recently suggested that they do crosstalk when are presented together and trigger effects that are not predicted from their individual functions. It is hypothesized indeed that crosstalk would increase the repertoire of responses of a given neuron during differentiation and explain why a relative limited number of these molecules is able to built a complex structure such as the nervous system. During this CIG, our studies have revealed the molecular mechanisms of how two of the most well studied axon guidance molecules, Netrin-1 and Slit1, cooperate during development to properly establish the topographic map of the thalamocortical projections (TCAs) (1,2). These projections connect the thalamus with the cortex and are important for processing of the sensory information from the environment. In particular, we found that FLRT3 interacts with the Slit1 receptor Robo1 and changes its repulsive signaling toward an up-regulation of the Netrin-1 receptor DCC which then binds to Netrin-1 and trigger attraction. In this context therefore, Slit1 acts a permissive cue for Netrin-1-induced attraction.
During this CIG, we also found that FLRTs, and in particular FLRT2 and FLRT3, cooperate in maintaining the interneuron migratory streams within the developing cortex. Although far less abundant, interneurons play an essential role in cortex excitability which is highlighted by the fact that defects in interneuron function often underlies neurological and psychiatric disorders including epilepsy and schizophrenia. Within the cortex, interneurons follow three main stereotyped routes of tangential migration: two very prominent located in the marginal zone (MZ) and between the intermediate zone (IZ) and the SVZ, and a third one, less abundant, located in the subplate (SP). Interestingly, these routes avoid FLRT expressing territories suggesting that FLRTs migt act as repulsive cues for cortical interneurons. The mechanisms and factors that maintain cortical interneuron streams and the final position of interneurons within the cortex are essentially unknown. We observed that double deletion of FLRT2 and FLRT3 in the whole nervous system of the mouse, affected specifically the migration of the SP stream interneurons and not those of the MZ and the IZ/SVZ (3). In particular, we observed a decrease in the number of SP stream interneurons concomitant with an increase of interneurons in deeper positions. The deletion of FLRT2 and FLRT3 did not affect the total number of interneurons reaching the cortex, indicating that subpalial migration was not affected and that the defect was related to the distribution of interneurons once they reached the cortex. Using the Emx1-Cre mouse line, we conditionally deleted FLRT2 and FLRT3 specifically from the cortex, without perturbing their expression in the migrating interneurons. These animals displayed exactly the same interneuron migration phenotype in the SP stream, indicating that FLRT2 and FLRT3 act non-cell autonomously, probably as ligands for an interneuron receptor. The only receptors that have been shown so far to bind to FLRTs are the Unc5 familly of transmembrane proteins that normally bind to Netrin-1 to trigger repulsion. Among these receptors, Unc5B and Unc5D are the ones which showed the higher affinity for FLRTs. At this point, we analyzed double knock-out brains for Unc5B and Unc5D but we did not detect any defect in intracortical interneuron migration, suggesting that FLRTs probably act trough different receptors.
As described above, FLRTs can act cell-autonomously and non-cell autonomously. During this CIG we became specifically interested in the intracellular mechanisms underlying the function of FLRT proteins in a cell-autonomous manner. In this context, previous reports suggested that the Rnd familly of Rho GTPases could interact with FLRTs and regulate cell adhesion and gastrulation in the Xenopus embryo. We addressed if such an interaction could also take place in neurons and if this interaction could regulate nervous system development. Co-immunoprecipitation and co-localization assays in heterologous cells as well as in neurons demonstrated that FLRTs and Rnd proteins could indeed interact. From these studies, we observed a strong interaction between FLRT3 and Rnd3 which we wanted to analyze in vivo using knock-out animals. The analysis of Rnd3 knock-outs revealed important axon guidance phenotypes in the developing brain, mainly affecting the developing of the TCAs. Tracing experiments demonstrated that TCAs, once they cross the diencephalon–telencephalon boundary (DTB) failed to proceed to the cortex and, instead, are found in ventral positions of the subpallium of the Rnd3 mutants. None of the FLRT mutants showed this severe phenotype suggesting that Rnd3 in this contex has functions that are not related with FLRTs. We are currently characterizing this phenotype, the molecular mechansims involved and to what extend some of the features of this phenotype depend on FLRT interaction (4).
Taken together we think that the results of this CIG provide important clues to the field of molecular neurobiology establishing FLRTs as important regulators of nervous system development and as potential therapeutic targets for neurological diseases. The role FLRTs in TCA projection, interneuron migration and Rnd interaction, have expanded previous data about FLRT function in excitatory neuron migration (5) and synapse formation (6) indicating that FLRTs are important multi-task players, not only because of the different processes they control but also because their different mechanisms of action, depending on the cellular context. More broadly, we think that our results have highlighted the relevance of transmembrane proteins with leucine-rich repeats in their extracellular domains as important molecular regulators of nervous system development. In this context, we have made an expression screening with other members of this family of proteins and we have observed that the majority of them display interesting patterns of expression in the developing brain of both, mice and humans (7).
Finally, the CIG was essential to establish an independent new molecular neurobiology laboratory in the Host Institution and to burst the research activity of the group to be promoted at the national and international levels. Recently, the Agencia Nacional de Evaluación y Prospectiva as well as the Agència de Qualitat del Sistema Universitari de Catalunya, two external and independent committees from the Spanish and Catalan governments, respectively, evaluated the research performance of my group and my personal research trajectory as outstanding. These positive evaluations are important because they are requirements for being eligible for a permanent position which the Host Institution has recently committed to promote within this year 2016 or next year, 2017. Finally, during this CIG I have been combining my research with other activities within the Host Institution which have improved my teaching skills.
1. Leyva-Diaz et al., Current Biology 2014 (contribution of this CIG)
2. Akita et al., J Physiol Sci 2015 (contribution of this CIG)
3. Fleitas et al., manuscript in preparation (contribution of this CIG)
4. Marfull et al., manuscript in preparation (contribution of this CIG)
5. Yamagishi et al., EMBO J 2011
6. O'Sullivan et al., Neuron 2012
7. Chauhan et al., manuscript in preparation (contribution of this CIG)
Traditionally, the effects of the axon guidance molecules and the intracellular mechanisms involved have been studied individually. However, it has been recently suggested that they do crosstalk when are presented together and trigger effects that are not predicted from their individual functions. It is hypothesized indeed that crosstalk would increase the repertoire of responses of a given neuron during differentiation and explain why a relative limited number of these molecules is able to built a complex structure such as the nervous system. During this CIG, our studies have revealed the molecular mechanisms of how two of the most well studied axon guidance molecules, Netrin-1 and Slit1, cooperate during development to properly establish the topographic map of the thalamocortical projections (TCAs) (1,2). These projections connect the thalamus with the cortex and are important for processing of the sensory information from the environment. In particular, we found that FLRT3 interacts with the Slit1 receptor Robo1 and changes its repulsive signaling toward an up-regulation of the Netrin-1 receptor DCC which then binds to Netrin-1 and trigger attraction. In this context therefore, Slit1 acts a permissive cue for Netrin-1-induced attraction.
During this CIG, we also found that FLRTs, and in particular FLRT2 and FLRT3, cooperate in maintaining the interneuron migratory streams within the developing cortex. Although far less abundant, interneurons play an essential role in cortex excitability which is highlighted by the fact that defects in interneuron function often underlies neurological and psychiatric disorders including epilepsy and schizophrenia. Within the cortex, interneurons follow three main stereotyped routes of tangential migration: two very prominent located in the marginal zone (MZ) and between the intermediate zone (IZ) and the SVZ, and a third one, less abundant, located in the subplate (SP). Interestingly, these routes avoid FLRT expressing territories suggesting that FLRTs migt act as repulsive cues for cortical interneurons. The mechanisms and factors that maintain cortical interneuron streams and the final position of interneurons within the cortex are essentially unknown. We observed that double deletion of FLRT2 and FLRT3 in the whole nervous system of the mouse, affected specifically the migration of the SP stream interneurons and not those of the MZ and the IZ/SVZ (3). In particular, we observed a decrease in the number of SP stream interneurons concomitant with an increase of interneurons in deeper positions. The deletion of FLRT2 and FLRT3 did not affect the total number of interneurons reaching the cortex, indicating that subpalial migration was not affected and that the defect was related to the distribution of interneurons once they reached the cortex. Using the Emx1-Cre mouse line, we conditionally deleted FLRT2 and FLRT3 specifically from the cortex, without perturbing their expression in the migrating interneurons. These animals displayed exactly the same interneuron migration phenotype in the SP stream, indicating that FLRT2 and FLRT3 act non-cell autonomously, probably as ligands for an interneuron receptor. The only receptors that have been shown so far to bind to FLRTs are the Unc5 familly of transmembrane proteins that normally bind to Netrin-1 to trigger repulsion. Among these receptors, Unc5B and Unc5D are the ones which showed the higher affinity for FLRTs. At this point, we analyzed double knock-out brains for Unc5B and Unc5D but we did not detect any defect in intracortical interneuron migration, suggesting that FLRTs probably act trough different receptors.
As described above, FLRTs can act cell-autonomously and non-cell autonomously. During this CIG we became specifically interested in the intracellular mechanisms underlying the function of FLRT proteins in a cell-autonomous manner. In this context, previous reports suggested that the Rnd familly of Rho GTPases could interact with FLRTs and regulate cell adhesion and gastrulation in the Xenopus embryo. We addressed if such an interaction could also take place in neurons and if this interaction could regulate nervous system development. Co-immunoprecipitation and co-localization assays in heterologous cells as well as in neurons demonstrated that FLRTs and Rnd proteins could indeed interact. From these studies, we observed a strong interaction between FLRT3 and Rnd3 which we wanted to analyze in vivo using knock-out animals. The analysis of Rnd3 knock-outs revealed important axon guidance phenotypes in the developing brain, mainly affecting the developing of the TCAs. Tracing experiments demonstrated that TCAs, once they cross the diencephalon–telencephalon boundary (DTB) failed to proceed to the cortex and, instead, are found in ventral positions of the subpallium of the Rnd3 mutants. None of the FLRT mutants showed this severe phenotype suggesting that Rnd3 in this contex has functions that are not related with FLRTs. We are currently characterizing this phenotype, the molecular mechansims involved and to what extend some of the features of this phenotype depend on FLRT interaction (4).
Taken together we think that the results of this CIG provide important clues to the field of molecular neurobiology establishing FLRTs as important regulators of nervous system development and as potential therapeutic targets for neurological diseases. The role FLRTs in TCA projection, interneuron migration and Rnd interaction, have expanded previous data about FLRT function in excitatory neuron migration (5) and synapse formation (6) indicating that FLRTs are important multi-task players, not only because of the different processes they control but also because their different mechanisms of action, depending on the cellular context. More broadly, we think that our results have highlighted the relevance of transmembrane proteins with leucine-rich repeats in their extracellular domains as important molecular regulators of nervous system development. In this context, we have made an expression screening with other members of this family of proteins and we have observed that the majority of them display interesting patterns of expression in the developing brain of both, mice and humans (7).
Finally, the CIG was essential to establish an independent new molecular neurobiology laboratory in the Host Institution and to burst the research activity of the group to be promoted at the national and international levels. Recently, the Agencia Nacional de Evaluación y Prospectiva as well as the Agència de Qualitat del Sistema Universitari de Catalunya, two external and independent committees from the Spanish and Catalan governments, respectively, evaluated the research performance of my group and my personal research trajectory as outstanding. These positive evaluations are important because they are requirements for being eligible for a permanent position which the Host Institution has recently committed to promote within this year 2016 or next year, 2017. Finally, during this CIG I have been combining my research with other activities within the Host Institution which have improved my teaching skills.
1. Leyva-Diaz et al., Current Biology 2014 (contribution of this CIG)
2. Akita et al., J Physiol Sci 2015 (contribution of this CIG)
3. Fleitas et al., manuscript in preparation (contribution of this CIG)
4. Marfull et al., manuscript in preparation (contribution of this CIG)
5. Yamagishi et al., EMBO J 2011
6. O'Sullivan et al., Neuron 2012
7. Chauhan et al., manuscript in preparation (contribution of this CIG)