Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

BACKGROUND: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. RESULTS: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo. CONCLUSIONS: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.

Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth.

Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-κB binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography.

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.

Slit2 activity in the migration of guidepost neurons shapes thalamic projections during development and evolution.

How brain connectivity has evolved to integrate the mammalian-specific neocortex remains largely unknown. Here, we address how dorsal thalamic axons, which constitute the main input to the neocortex, are directed internally to their evolutionary novel target in mammals, though they follow an external path to other targets in reptiles and birds. Using comparative studies and functional experiments in chick, we show that local species-specific differences in the migration of previously identified "corridor" guidepost neurons control the opening of a mammalian thalamocortical route. Using in vivo and ex vivo experiments in mice, we further demonstrate that the midline repellent Slit2 orients migration of corridor neurons and thereby switches thalamic axons from an external to a mammalian-specific internal path. Our study reveals that subtle differences in the migration of conserved intermediate target neurons trigger large-scale changes in thalamic connectivity, and opens perspectives on Slit functions and the evolution of brain wiring.

Topography of thalamic projections requires attractive and repulsive functions of Netrin-1 in the ventral telencephalon.

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A-C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1-rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1-rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.

Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain.

The function of the nervous system depends on the precision of axon wiring during development. Previous studies have demonstrated that Slits, a family of secreted chemorepellent proteins, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 or, even more so, in both Slit1 and Slit2 have defects in multiple axonal pathways, including corticofugal, thalamocortical, and callosal connections. In the spinal cord, members of the Robo family of proteins help mediate the function of Slits, but the relative contribution of these receptors to the guidance of forebrain projections remains to be determined. In the present study, we addressed the function of Robo1 and Robo2 in the guidance of forebrain projections by analyzing Robo1-, Robo2-, and Robo1;Robo2-deficient mice. Mice deficient in Robo2 and, more dramatically, in both Robo1 and Robo2, display prominent axon guidance errors in the development of corticofugal, thalamocortical, and corticocortical callosal connections. Our results demonstrate that Robo1 and Robo2 mostly cooperate to mediate the function of Slit proteins in guiding the major forebrain projections.

Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain.

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.