Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons.

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

Remotely Produced and Axon-Derived Netrin-1 Instructs GABAergic Neuron Migration and Dopaminergic Substantia Nigra Development.

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviors and show select disease vulnerability, including in Parkinson's disease. Despite progress in identifying mDA neuron subtypes, how these neuronal subsets develop and organize into functional brain structures remains poorly understood. Here we generate and use an intersectional genetic platform, Pitx3-ITC, to dissect the mechanisms of substantia nigra (SN) development and implicate the guidance molecule Netrin-1 in the migration and positioning of mDA neuron subtypes in the SN. Unexpectedly, we show that Netrin-1, produced in the forebrain and provided to the midbrain through axon projections, instructs the migration of GABAergic neurons into the ventral SN. This migration is required to confine mDA neurons to the dorsal SN. These data demonstrate that neuron migration can be controlled by remotely produced and axon-derived secreted guidance cues, a principle that is likely to apply more generally.

Mesencephalic origin of the rostral Substantia nigra pars reticulata.

In embryonic development, the neurons that will constitute a heterogeneous nucleus may have distinct origins. The different components of these populations reach their final location by radial and tangential migrations. The Substantia nigra pars reticulata (SNR) presents a high level of neuronal heterogeneity. It is composed by GABAergic neurons located in the mes-diencephalic basal plate. These inhibitory neurons usually display tangential migrations and it has been already described that the caudal SNR is colonized tangentially from rhombomere 1. Our aim is to unveil the origin of the rostral SNR. We have localized a Nkx6.2 positive ventricular domain located in the alar midbrain. Nkx6.2 derivatives' fate map analysis showed mainly a rostral colonization of this GABAergic neuronal population. We confirmed the mesencephalic origin by the expression of Six3. Both transcription factors are sequentially expressed along the differentiation of these neurons. We demonstrated the origin of the rostral SNR; our data allowed us to postulate that this nucleus is composed by two neuronal populations distributed in opposite gradients with different origins, one from rhombomere 1, caudal to rostral, and the other from the midbrain, rostral to caudal. We can conclude that the SNR has multiple origins and follows complex mechanisms of specification and migration. Our results support vital information for the study of genetic modifications in these extremely complex processes that result in devastating behavioral alterations and predisposition to psychiatric diseases. Understanding the development, molecular identity and functional characteristics of these diverse neuronal populations might lead to better diagnosis and treatment of several forms of neurological and psychiatric disease.

Developmental guidance of the retroflex tract at its bending point involves Robo1-Slit2-mediated floor plate repulsion.

The retroflex tract contains medial habenula efferents that target the hindbrain interpeduncular complex and surrounding areas. This tract displays a singular course. Initially, habenular axons extend ventralwards in front of the pretectum until they reach the basal plate. Next, they avoid crossing the local floor plate, sharply changing course caudalwards (the retroflexion alluded by the tract name) and navigate strictly antero-posteriorly across basal pretectum, midbrain and isthmus. Once they reach rhombomere 1, the habenular axons criss-cross the floor plate several times within the interpeduncular nuclear complex as they innervate it. Here we described the timing and details of growth phenomena as these axons navigate to their target. The first dorsoventral course apparently obeys Ntn1 attraction. We checked the role of local floor plate signaling in the decision to avoid the thalamic floor plate and bend caudalwards. Analyzing the altered floor and basal plates of Gli2 knockout mice, we found a contralateral projection of most habenular axons, plus ulterior bizarre navigation rostralwards. This crossing phenotype was due to a reduced expression of Slit repulsive cues, suggesting involvement of the floor-derived Robo-Slit system in the normal guidance of this tract. Using Slit and Robo mutant mice, open neural tube and co-culture assays, we determined that Robo1-Slit2 interaction is specifically required for impeding that medial habenular axons cross the thalamic floor plate. This pathfinding mechanism is essential to establish the functionally important habenulo-interpeduncular connection.

Mesencephalic basolateral domain specification is dependent on Sonic Hedgehog.

In the study of central nervous system morphogenesis, the identification of new molecular markers allows us to identify domains along the antero-posterior and dorso-ventral (DV) axes. In the past years, the alar and basal plates of the midbrain have been divided into different domains. The precise location of the alar-basal boundary is still under discussion. We have identified Barhl1, Nhlh1 and Six3 as appropriate molecular markers to the adjacent domains of this transition. The description of their expression patterns and the contribution to the different mesencephalic populations corroborated their role in the specification of these domains. We studied the influence of Sonic Hedgehog on these markers and therefore on the specification of these territories. The lack of this morphogen produced severe alterations in the expression pattern of Barhl1 and Nhlh1 with consequent misspecification of the basolateral (BL) domain. Six3 expression was apparently unaffected, however its distribution changed leading to altered basal domains. In this study we confirmed the localization of the alar-basal boundary dorsal to the BL domain and demonstrated that the development of the BL domain highly depends on Shh.

Red nucleus and rubrospinal tract disorganization in the absence of Pou4f1.

The red nucleus (RN) is a neuronal population that plays an important role in forelimb motor control and locomotion. Histologically it is subdivided into two subpopulations, the parvocellular RN (pRN) located in the diencephalon and the magnocellular RN (mRN) in the mesencephalon. The RN integrates signals from motor cortex and cerebellum and projects to spinal cord interneurons and motor neurons through the rubrospinal tract (RST). Pou4f1 is a transcription factor highly expressed in this nucleus that has been related to its specification. Here we profoundly analyzed consequences of Pou4f1 loss-of-function in development, maturation and axonal projection of the RN. Surprisingly, RN neurons are specified and maintained in the mutant, no cell death was detected. Nevertheless, the nucleus appeared disorganized with a strong delay in radial migration and with a wider neuronal distribution; the neurons did not form a compacted population as they do in controls, Robo1 and Slit2 were miss-expressed. Cplx1 and Npas1, expressed in the RN, are transcription factors involved in neurotransmitter release, neuronal maturation and motor function processes among others. In our mutant mice, both transcription factors are lost, suggesting an abnormal maturation of the RN. The resulting altered nucleus occupied a wider territory. Finally, we examined RST development and found that the RN neurons were able to project to the spinal cord but their axons appeared defasciculated. These data suggest that Pou4f1 is necessary for the maturation of RN neurons but not for their specification and maintenance.