Single-cell landscapes of primary glioblastomas and matched explants and cell lines show variable retention of inter- and intratumor heterogeneity.

Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.

Neurog2 Acts as a Classical Proneural Gene in the Ventromedial Hypothalamus and Is Required for the Early Phase of Neurogenesis.

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.

Ascl1 is required to specify a subset of ventromedial hypothalamic neurons.

Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.

Oligodendrocyte development in the embryonic tuberal hypothalamus and the influence of Ascl1.

BACKGROUND: Although the vast majority of cells in our brains are glia, we are only beginning to understand programs governing their development, especially within the embryonic hypothalamus. In mice, gliogenesis is a protracted process that begins during embryonic stages and continues into the early postnatal period, with glial progenitors first producing oligodendrocyte precursor cells, which then differentiate into pro-oligodendrocytes, pro-myelinating oligodendrocytes, and finally, mature myelinating oligodendrocytes. The exact timing of the transition from neurogenesis to gliogenesis and the subsequent differentiation of glial lineages remains unknown for most of the Central Nervous System (CNS), and is especially true for the hypothalamus. METHODS: Here we used mouse embryonic brain samples to determine the onset of gliogenesis and expansion of glial populations in the tuberal hypothalamus using glial markers Sox9, Sox10, Olig2, PdgfRα, Aldh1L1, and MBP. We further employed Ascl1 and Neurog2 mutant mice to probe the influence of these proneual genes on developing embryonic gliogenic populations. RESULTS: Using marker analyses for glial precursors, we found that gliogenesis commences just prior to E13.5 in the tuberal hypothalamus, beginning with the detection of glioblast and oligodendrocyte precursor cell markers in a restricted domain adjacent to the third ventricle. Sox9+ and Olig2+ glioblasts are also observed in the mantle region from E13.5 onwards, many of which are Ki67+ proliferating cells, and peaks at E17.5. Using Ascl1 and Neurog2 mutant mice to investigate the influence of these bHLH transcription factors on the progression of gliogenesis in the tuberal hypothalamus, we found that the elimination of Ascl1 resulted in an increase in oligodendrocyte cells throughout the expansive period of oligodendrogenesis. CONCLUSION: Our results are the first to define the timing of gliogenesis in the tuberal hypothalamus and indicate that Ascl1 is required to repress oligodendrocyte differentiation within this brain region.