Andrea Faedo

Cortical regions are determined by patterning transcription factors and segregated by differential cell affinity.

Development of the human striatum revealed: Deep in the brain, the striatum receives and coordinates inputs from other parts of the brain. Bocchi et al. surveyed molecular features as the striatum develops in the human brain. Single-cell surveys of long intergenic noncoding RNAs revealed a progenitor for medium spiny neurons and provide insight into evolutionary divergence of this critical part of the brain. Science , this issue p. eabf5759

DACH1 is the human homolog of the Drosophila dachshund gene, which is involved in the development of the eye, nervous system, and limbs in the fly. Here, we systematically investigate DACH1 expression patterns during human neurodevelopment, from 5 to 21 postconceptional weeks. By immunodetection analysis, we found that DACH1 is highly expressed in the proliferating neuroprogenitors of the developing cortical ventricular and subventricular regions, while it is absent in the more differentiated cortical plate. Single-cell global transcriptional analysis revealed that DACH1 is specifically enriched in neuroepithelial and ventricular radial glia cells of the developing human neocortex. Moreover, we describe a previously unreported DACH1 expression in the human striatum, in particular in the striatal medium spiny neurons. This finding qualifies DACH1 as a new striatal projection neuron marker, together with PPP1R1B, BCL11B, and EBF1. We finally compared DACH1 expression profile in human and mouse forebrain, where we observed spatio-temporal similarities in its expression pattern thus providing a precise developmental description of DACH1 in the 2 mammalian species.

Significance: We report that huntingtin mutation affects specific aspects of human neurodevelopment at the level of neuronal progenitor specification and its early commitment, leading to an abnormal cell organization and acquisition of mature neuronal identities in cerebral organoids. We also show that down-regulation of mutant huntingtin or pharmacological inhibition of one of its effectors, ADAM10, successfully rescues neuronal differentiation, suggesting that an early intervention may revert neurodegeneration later in life.

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington9s disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance. Significance We established human embryonic stem (hES) cell-inducible lines to express specific transcription factors [GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1)] to improve medium spiny neuron (MSN) differentiation and to study human striatal development in vitro. We also used a nonintegrating system, by utilizing modified mRNAs to transiently overexpress Gsx2 and Ebf1 in a different hES cell line. These data can help to improve the differentiation protocols that aim to produce high-quality cell preparation suitable for cell transplantation in animal models of Huntington’s disease. Finally, we show that hES-derived striatal progenitors can be transplanted in the striatum of animal models and can differentiate and integrate into the host, extending fibers over a long distance (including the substantia nigra, a striatal target).

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a “bivalent” histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.

Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington’s Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1⁺/GSX2⁺ progenitor cells of the lateral ganglionic eminence (LGE) at day 15–25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2⁺/GABA⁺ neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs. The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

We examined the role of the orphan nuclear hormone receptor CoupTFI in mediating cortical development downstream of meningeal retinoic acid signaling. CoupTFI is a regulator of cortical development known to collaborate with retinoic acid (RA) signaling in other systems. To examine the interaction of CoupTFI and cortical RA signaling we utilized Foxc1-mutant mice in which defects in meningeal development lead to alterations in cortical development due to a reduction of RA signaling. By analyzing CoupTFI^−/−;Foxc1^H/L double mutant mice we provide evidence that CoupTFI is required for RA rescue of the ventricular zone and the neurogenic phenotypes in Foxc1-mutants. We also found that overexpression of CoupTFI in Foxc1-mutants is sufficient to rescue the Foxc1-mutant cortical phenotype in part. These results suggest that CoupTFI collaborates with RA signaling to regulate both cortical ventricular zone progenitor cell behavior and cortical neurogenesis.

Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington’s disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFβ inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1⁺/GSX2⁺ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1⁺/FOXP2⁺/CTIP2⁺/calbindin⁺/DARPP-32⁺ MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32⁺ neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.