Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome

Defects in interneuron migration during forebrain development can disrupt the assembly of cortical circuits and have been associated with neuropsychiatric disease. The molecular and cellular bases of such deficits have been particularly difficult to study in humans due to limited access to functional forebrain tissue from patients. We previously developed a human forebrain assembloid model of Timothy Syndrome (TS), caused by a gain-of-function mutation in CACNA1C which encodes the L-type calcium channel (LTCC) Cav1.2. By functionally integrating human induced pluripotent stem cell (hiPSC)-derived organoids resembling the dorsal and ventral forebrain from patients and control individuals, we uncovered that migration is disrupted in TS cortical interneurons. Here, we dissect the molecular underpinnings of this phenotype and report that acute pharmacological modulation of Cav1.2 can rescue the saltation length but not the saltation frequency of TS migrating interneurons. Furthermore, we find that the defect in saltation length in TS interneurons is associated with aberrant actomyosin function and is rescued by pharmacological modulation of MLC phosphorylation, whereas the saltation frequency phenotype in TS interneurons is driven by enhanced GABA sensitivity and can be restored by GABA receptor antagonism. Overall, these findings uncover multi-faceted roles of LTCC function in human cortical interneuron migration in the context of disease and suggest new strategies to restore interneuron migration deficits.

Semaphorin 3C attracts MGE-derived cortical interneurons in the deep migratory stream guiding them into the developing neocortex

While it is known that Semaphorin 3C acts as a guidance cue for axons during brain development, their potential role during interneuron migration is largely unknown. One striking observation is that Sema3C demarcates the pallial/subpallial border and the intracortical pathway of cortical interneurons in the dorsal telencephalon. Moreover, migrating cortical interneurons express Neuropilin1 and Neuropilin2, described receptors for Semaphorin 3A, 3F and 3C. All these reasons prompt us to examine possible roles for Sema3C on cortical interneuron migration. Using several in vitro approaches, we showed that Nrp1-expressing MGE-derived interneurons from the deep migratory stream migrate towards the increasing Sema3C gradients. In contrast, inhibitory neurons from the superficial migratory stream that express Nrp2, do not respond to this guidance cue. In the present study, we proposed that diffusible Sema3C expressed in the Pallium provides a permissive corridor that attracts the Nrp1- expressing interneurons from the DMS into the dorsal telencephalon.

Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons

GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb. Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2, Nkx2-2, and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.