During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5–E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

Class 3 Semaphorins are a subfamily of chemotropic molecules implicated in the projection of dopaminergic neurons from the ventral mesencephalon and in the formation of the nigrostriatal pathway (NSP) during embryonic development. In humans, loss of mesencephalic dopaminergic neurons leads to Parkinson's disease (PD). Cell replacement therapy with dopaminergic neurons generated from embryonic stem cells (ES-TH + ) is being actively explored in models of PD. Among several requisites for this approach to work are the adequate reconstruction of the NSP and the correct innervation of normal striatal targets by dopaminergic axons. In this work, we characterized the response of ES-TH + neurons to Semaphorins 3A, 3C, and 3F, and compared it with that of tyrosine hidroxylase-positive neurons (TH + ) obtained from embryonic ventral mesencephalon (VM-TH + ). We observed that similar proportions of ES-TH + and VM-TH + neurons express Semaphorin receptors Neuropilin 1 and 2. Furthermore, the axons of both populations responded very similarly to Semaphorin exposure: Semaphorin 3A increased axon length, and Semaphorin 3C attracted axons and increased their length. These effects were mediated by Neuropilins, since addition of blocking antibodies against these proteins reduced the effects on axonal growth and attraction, and only TH + axons expressing Neuropilins responded to the Semaphorins analyzed. The observations reported here show phenotypic similarities between VM-TH + and ES-TH + neurons, and suggest that Semaphorins 3A and 3C could be employed to guide axons of grafted ES-TH + in therapeutic protocols for PD.

Cell therapy in animal models of Parkinson's disease (PD) is effective after intrastriatal grafting of dopamine (DA) neurons, whereas intranigral transplantation of dopaminergic cells does not cause consistent behavioral recovery. One strategy to promote axonal growth of dopaminergic neurons from the substantia nigra (SN) to the striatum is degradation of inhibitory components such as chondroitin sulphate proteoglycans (CSPG). An alternative is the guidance of DA axons by chemotropic agents. Semaphorins 3A and 3C enhance axonal growth of embryonic stem (ES) cell-derived dopaminergic neurons in vitro, while Semaphorin 3C also attracts them. We asked whether intranigral transplantation of DA neurons, combined with either degradation of CSPG or with grafts of Semaphorin 3-expressing cells, towards the striatum, is effective in establishing a new nigrostriatal dopaminergic pathway in rats with unilateral depletion of DA neurons. We found depolarization-induced DA release in dorsal striatum, DA axonal projections from SN to striatum, and concomitant behavioral improvement in Semaphorin 3-treated animals. These effects were absent in animals that received intranigral transplants combined with Chondroitinase ABC treatment, although partial degradation of CSPG was observed. These results are evidence that Semaphorin 3-directed long-distance axonal growth of dopaminergic neurons, resulting in behavioral improvement, is possible in adult diseased brains.

The Nigrostriatal pathway (NSP) is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

Stereotypic cell migrations in the developing brain are fundamental for the proper patterning of brain regions and formation of neural networks. In this work, we uncovered in the developing rat, a population of neurons expressing tyrosine hydroxylase (TH) that migrates posteriorly from the alar plate of the midbrain, in neurophilic interaction with axons of the mesencephalic nucleus of the trigeminal nerve. A fraction of this population was also shown to traverse the mid-hindbrain boundary, reaching the vicinity of the locus coeruleus (LC) in rhombomere 1 (r1). This migratory population, however, does not have a noradrenergic (NA) phenotype and, in keeping with its midbrain origin, expresses Otx2 which is down regulated upon migration into the hindbrain. The interaction with the trigeminal mesencephalic axons is necessary for the arrangement and distribution of migratory cells as these aspects are dramatically altered in whole embryo cultures upon disruption of trigeminal axon projection by interfering with DCC function. Moreover, in mouse embryos in an equivalent developmental stage, we detected a cell population that also migrates caudally within the midbrain apposed to mesencephalic trigeminal axons but that does not express TH; a fraction of this population expresses calbindin instead. Overall, our work identified TH-expressing neurons from the rat midbrain alar plate that migrate tangentially over long distances within the midbrain and into the hindbrain by means of a close interaction with trigeminal mesencephalic axons. A different migratory population in this region and also in mouse embryos revealed diversity among the cells that follow this descending migratory pathway.