Transcriptomic atlas of midbrain dopamine neurons uncovers differential vulnerability in a Parkinsonism lesion model.

Midbrain dopamine (mDA) neurons comprise diverse cells with unique innervation targets and functions. This is illustrated by the selective sensitivity of mDA neurons of the substantia nigra compacta (SNc) in patients with Parkinson's disease, while those in the ventral tegmental area (VTA) are relatively spared. Here, we used single nuclei RNA sequencing (snRNA-seq) of approximately 70,000 mouse midbrain cells to build a high-resolution atlas of mouse mDA neuron diversity at the molecular level. The results showed that differences between mDA neuron groups could best be understood as a continuum without sharp differences between subtypes. Thus, we assigned mDA neurons to several 'territories' and 'neighborhoods' within a shifting gene expression landscape where boundaries are gradual rather than discrete. Based on the enriched gene expression patterns of these territories and neighborhoods, we were able to localize them in the adult mouse midbrain. Moreover, because the underlying mechanisms for the variable sensitivities of diverse mDA neurons to pathological insults are not well understood, we analyzed surviving neurons after partial 6-hydroxydopamine (6-OHDA) lesions to unravel gene expression patterns that correlate with mDA neuron vulnerability and resilience. Together, this atlas provides a basis for further studies on the neurophysiological role of mDA neurons in health and disease.

Schwann cell precursors represent a neural crest-like state with biased multipotency.

Schwann cell precursors (SCPs) are nerve-associated progenitors that can generate myelinating and non-myelinating Schwann cells but also are multipotent like the neural crest cells from which they originate. SCPs are omnipresent along outgrowing peripheral nerves throughout the body of vertebrate embryos. By using single-cell transcriptomics to generate a gene expression atlas of the entire neural crest lineage, we show that early SCPs and late migratory crest cells have similar transcriptional profiles characterised by a multipotent "hub" state containing cells biased towards traditional neural crest fates. SCPs keep diverging from the neural crest after being primed towards terminal Schwann cells and other fates, with different subtypes residing in distinct anatomical locations. Functional experiments using CRISPR-Cas9 loss-of-function further show that knockout of the common "hub" gene Sox8 causes defects in neural crest-derived cells along peripheral nerves by facilitating differentiation of SCPs towards sympathoadrenal fates. Finally, specific tumour populations found in melanoma, neurofibroma and neuroblastoma map to different stages of SCP/Schwann cell development. Overall, SCPs resemble migrating neural crest cells that maintain multipotency and become transcriptionally primed towards distinct lineages.

Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM-sleep regulatory neurons.

The midbrain reticular formation (MRF) is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including sleep regulation. However, the molecular characteristics and development of MRF neurons are poorly understood. As the transcription factor, Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is required for the expression of several GABAergic lineage-specific transcription factors, including Nkx2-2 and Skor2, which are co-expressed in a restricted group of post-mitotic GABAergic precursors in the MRF. Both Gata2 and Nkx2-2 function is required for Skor2 expression in GABAergic precursors. In the adult mouse and rat midbrain, Nkx2-2-and Skor2-expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the MRF, an area containing REM-off neurons regulating REM sleep. In addition to the characteristic localization, Skor2+ cells increase their activity upon REM-sleep inhibition, send projections to the dorsolateral pons, a region associated with sleep control, and are responsive to orexins, consistent with the known properties of midbrain REM-off neurons.

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation.

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development.

Midbrain dopamine (mDA) neurons constitute a heterogenous group of cells that have been intensely studied, not least because their degeneration causes major symptoms in Parkinson's disease. Understanding the diversity of mDA neurons - previously well characterized anatomically - requires a systematic molecular classification at the genome-wide gene expression level. Here, we use single cell RNA sequencing of isolated mouse neurons expressing the transcription factor Pitx3, a marker for mDA neurons. Analyses include cells isolated during development up until adulthood and the results are validated by histological characterization of newly identified markers. This identifies seven neuron subgroups divided in two major branches of developing Pitx3-expressing neurons. Five of them express dopaminergic markers, while two express glutamatergic and GABAergic markers, respectively. Analysis also indicate evolutionary conservation of diversity in humans. This comprehensive molecular characterization will provide a valuable resource for elucidating mDA neuron subgroup development and function in the mammalian brain.

Early prenatal alcohol exposure alters imprinted gene expression in placenta and embryo in a mouse model.

Prenatal alcohol exposure (PAE) can harm the embryonic development and cause life-long consequences in offspring's health. To clarify the molecular mechanisms of PAE we have used a mouse model of early alcohol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first eight days of gestation (GD 0.5-8.5). Owing to the detected postnatal growth-restricted phenotype in the offspring of this mouse model and both prenatal and postnatal growth restriction in alcohol-exposed humans, we focused on imprinted genes Insulin-like growth factor 2 (Igf2), H19, Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn) and Paternally expressed gene 3 (Peg3), which all are known to be involved in embryonic and placental growth and development. We studied the effects of alcohol on DNA methylation level at the Igf2/H19 imprinting control region (ICR), Igf2 differentially methylated region 1, Snrpn ICR and Peg3 ICR in 9.5 embryonic days old (E9.5) embryos and placentas by using MassARRAY EpiTYPER. To determine alcohol-induced alterations globally, we also examined methylation in long interspersed nuclear elements (Line-1) in E9.5 placentas. We did not observe any significant alcohol-induced changes in DNA methylation levels. We explored effects of PAE on gene expression of E9.5 embryos as well as E9.5 and E16.5 placentas by using quantitative PCR. The expression of growth promoter gene Igf2 was decreased in the alcohol-exposed E9.5 and E16.5 placentas. The expression of negative growth controller H19 was significantly increased in the alcohol-exposed E9.5 embryos compared to controls, and conversely, a trend of decreased expression in alcohol-exposed E9.5 and E16.5 placentas were observed. Furthermore, increased Snrpn expression in alcohol-exposed E9.5 embryos was also detected. Our study indicates that albeit no alterations in the DNA methylation levels of studied sequences were detected by EpiTYPER, early PAE can affect the expression of imprinted genes in both developing embryo and placenta.

Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages.

Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

A recessive syndrome of intellectual disability, moderate overgrowth, and renal dysplasia predisposing to Wilms tumor is caused by a mutation in FIBP gene.

Clinical classification of overgrowth syndromes represents a challenge since a wide spectrum of disorders result in marked overgrowth. Therefore, there is a continuous effort to identify the genetic basis of these disorders that will eventually facilitate their molecular classification. Here, we have identified the genetic etiology and the pathogenetic mechanism underlying a rare autosomal recessive overgrowth syndrome in three affected siblings. The overgrowth phenotype in the patients was accompanied by developmental delay, learning disabilities, and variable congenital abnormalities. To elucidate the genetic etiology of the disorder, whole-genome genotyping and whole-exome sequencing were used. The disease was mapped to 3p21.1-p14.2 and 11q13.1-q13.4, where an in-frame insertion (c.175_176insTAA) in FIBP gene was revealed. The resulting indel (p.H59LN) was predicted to change the protein conformation with likely deleterious effect on its function as one of the fibroblast growth factor signaling mediators. In vitro cellular proliferation assay and in situ hypridization in vivo were then performed to understand the pathophysiology of the disease. The patients' skin fibroblasts showed an increased proliferation capacity compared to the controls' explaining the observed overgrowth phenotype. In addition, we detected Fibp expression most notably in the brains of mice embryos suggesting a possible effect on cognitive functions early in development. To date, only one patient has been reported with a homozygous nonsense mutation in FIBP exhibiting an overgrowth syndrome with multiple congenital abnormalities. Taken all together, these findings provide convincing evidence implicating FIBP aberrations in the newly recognized overgrowth syndrome and expand the associated phenotypes to include possible Wilms tumor predisposition. © 2016 Wiley Periodicals, Inc.

Differentiation and molecular heterogeneity of inhibitory and excitatory neurons associated with midbrain dopaminergic nuclei.

Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits.

The role of Tal2 and Tal1 in the differentiation of midbrain GABAergic neuron precursors.

Midbrain- and hindbrain-derived GABAergic interneurons are critical for regulation of sleep, respiratory, sensory-motor and motivational processes, and they are implicated in human neurological disorders. However, the precise mechanisms that underlie generation of GABAergic neuron diversity in the midbrain-hindbrain region are poorly understood. Here, we show unique and overlapping requirements for the related bHLH proteins Tal1 and Tal2 in GABAergic neurogenesis in the midbrain. We show that Tal2 and Tal1 are specifically and sequentially activated during midbrain GABAergic neurogenesis. Similar to Gata2, a post-mitotic selector of the midbrain GABAergic neuron identity, Tal2 expression is activated very early during GABAergic neuron differentiation. Although the expression of Tal2 and Gata2 genes are independent of each other, Tal2 is important for normal midbrain GABAergic neurogenesis, possibly as a partner of Gata2. In the absence of Tal2, the majority of midbrain GABAergic neurons switch to a glutamatergic-like phenotype. In contrast, Tal1 expression is activated in a Gata2 and Tal2 dependent fashion in the more mature midbrain GABAergic neuron precursors, but Tal1 alone is not required for GABAergic neuron differentiation from the midbrain neuroepithelium. However, inactivation of both Tal2 and Tal1 in the developing midbrain suggests that the two factors co-operate to guide GABAergic neuron differentiation in a specific ventro-lateral midbrain domain. The observed similarities and differences between Tal1/Tal2 and Gata2 mutants suggest both co-operative and unique roles for these factors in determination of midbrain GABAergic neuron identities.

Distinct developmental origins and regulatory mechanisms for GABAergic neurons associated with dopaminergic nuclei in the ventral mesodiencephalic region.

GABAergic neurons in the ventral mesodiencephalic region are highly important for the function of dopaminergic pathways that regulate multiple aspects of behavior. However, development of these neurons is poorly understood. We recently showed that molecular regulation of differentiation of the GABAergic neurons associated with the dopaminergic nuclei in the ventral midbrain (VTA and SNpr) is distinct from the rest of midbrain, but the reason for this difference remained elusive. Here, we have analyzed the developmental origin of the VTA and SNpr GABAergic neurons by genetic fate mapping. We demonstrate that the majority of these GABAergic neurons originate outside the midbrain, from rhombomere 1, and move into the ventral midbrain only as postmitotic neuronal precursors. We further show that Gata2, Gata3 and Tal1 define a subpopulation of GABAergic precursors in ventral rhombomere 1. A failure in GABAergic neuron differentiation in this region correlates with loss of VTA and SNpr GABAergic neurons in Tal1 mutant mice. In contrast to midbrain, GABAergic neurons of the anterior SNpr in the diencephalon are not derived from the rhombomere 1. These results suggest unique migratory pathways for the precursors of important GABAergic neuron subpopulations, and provide the basis for understanding diversity within midbrain GABAergic neurons.

Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain.

The structure and projection patterns of adult mesodiencephalic dopaminergic (DA) neurons are one of the best characterized systems in the vertebrate brain. However, the early organization and development of these nuclei remain poorly understood. The induction of midbrain DA neurons requires sonic hedgehog (Shh) from the floor plate and fibroblast growth factor 8 (FGF8) from the isthmic organizer, but the way in which FGF8 regulates DA neuron development is unclear. We show that, during early embryogenesis, mesodiencephalic neurons consist of two distinct populations: a diencephalic domain, which is probably independent of isthmic FGFs; and a midbrain domain, which is dependent on FGFs. Within these domains, DA progenitors and precursors use partly different genetic programs. Furthermore, the diencephalic DA domain forms a distinct cell population, which also contains non-DA Pou4f1(+) cells. FGF signaling operates in proliferative midbrain DA progenitors, but is absent in postmitotic DA precursors. The loss of FGFR1/2-mediated signaling results in a maturation failure of the midbrain DA neurons and altered patterning of the midbrain floor. In FGFR mutants, the DA domain adopts characteristics that are typical for embryonic diencephalon, including the presence of Pou4f1(+) cells among TH(+) cells, and downregulation of genes typical of midbrain DA precursors. Finally, analyses of chimeric embryos indicate that FGF signaling regulates the development of the ventral midbrain cell autonomously.

FGF signaling gradient maintains symmetrical proliferative divisions of midbrain neuronal progenitors.

For the correct development of the central nervous system, the balance between self-renewing and differentiating divisions of the neuronal progenitors must be tightly regulated. To maintain their self-renewing identity, the progenitors need to retain both apical and basal interfaces. However, the identities of fate-determining signals which cells receive via these connections, and the exact mechanism of their action, are poorly understood. The conditional inactivation of Fibroblast growth factor (FGF) receptors 1 and 2 in the embryonic mouse midbrain-hindbrain area results in premature neuronal differentiation. Here, we aim to elucidate the connection between FGF signaling and neuronal progenitor maintenance. Our results reveal that the loss of FGF signaling leads to downregulation of Hes1 and upregulation of Ngn2, Dll1, and p57 in the ventricular zone (VZ) cells, and that this increased neurogenesis occurs cell-autonomously. Yet the cell cycle progression, apico-basal-polarity, cell-cell connections, and the positioning of mitotic spindle in the mutant VZ appear unaltered. Interestingly, FGF8-protein is highly concentrated in the basal lamina. Thus, FGFs may act through basal processes of neuronal progenitors to maintain their progenitor status. Indeed, midbrain neuronal progenitors deprived in vitro of FGFs switched from symmetrical proliferative towards symmetrical neurogenic divisions. We suggest that FGF signaling in the midbrain VZ is cell-autonomously required for the maintenance of symmetrical proliferative divisions via Hes1-mediated repression of neurogenic genes.

Fibroblast growth factor receptors cooperate to regulate neural progenitor properties in the developing midbrain and hindbrain.

Fibroblast growth factors (FGFs) secreted from the midbrain-rhombomere 1 (r1) boundary instruct cell behavior in the surrounding neuroectoderm. For example, a combination of FGF and sonic hedgehog (SHH) can induce the development of the midbrain dopaminergic neurons, but the mechanisms behind the action and integration of these signals are unclear. We studied how FGF receptors (FGFRs) regulate cellular responses by analyzing midbrain-r1 development in mouse embryos, which carry different combinations of mutant Fgfr1, Fgfr2, and Fgfr3 alleles. Our results show that the FGFRs act redundantly to support cell survival in the dorsal neuroectoderm, promote r1 tissue identity, and regulate the production of ventral neuronal populations, including midbrain dopaminergic neurons. The compound Fgfr mutants have apparently normal WNT/SHH signaling and neurogenic gene expression in the ventral midbrain, but the number of proliferative neural progenitors is reduced as a result of precocious neuronal differentiation. Our results suggest a SoxB1 family member, Sox3, as a potential FGF-induced transcription factor promoting progenitor renewal. We propose a model for regulation of progenitor cell self-renewal and neuronal differentiation by combinatorial intercellular signals in the ventral midbrain.

FGF regulated gene-expression and neuronal differentiation in the developing midbrain-hindbrain region.

The neuroectodermal tissue close to the midbrain-hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) secretes signaling molecules, such as fibroblast growth factors (FGFs), which regulate cellular survival, patterning and proliferation in the midbrain and rhombomere 1 (R1) of the hindbrain. We have previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. Here, we have compared the gene expression profiles of midbrain-R1 tissues from wild-type embryos and conditional Fgfr1 mutants, in which FGFR1 is inactivated in the midbrain and R1. Loss of Fgfr1 results in the downregulation of several genes expressed close to the midbrain-hindbrain boundary and in the disappearance of gene expression gradients in the midbrain and anterior hindbrain. Our screen identified several previously uncharacterized genes which may participate in the development of midbrain-R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1.

Linkage to two separate loci in a family with a novel distal myopathy phenotype (MPD3).

We recently described a new type of adult onset distal myopathy (MPD3) with autosomal dominant inheritance. The onset of symptoms is around the age of 30 and the characteristic first symptoms include clumsiness of the hands and stumbling. The thenar and hypothenar muscles are involved at the onset. The disease progressed to the intrinsic muscles of the hands, both anterior and posterior muscle compartments of the lower legs, the forearm muscles, and later to the proximal muscles. Dystrophic changes with rimmed vacuoles were observed in the muscle biopsy. We have performed a genome wide scan here in order to identify the MPD3 locus. Unexpectedly, markers on two distinct chromosomal regions 8p22-q11 and 12q13-q22, provided significant evidence for linkage in this family. Multipoint linkage analyses produced equal maximum multipoint LOD score of 3.01 for both chromosomal regions and haplotype analysis showed a specific haplotype segregating with the disease for both loci. It is thus impossible to distinguish between two loci without additional family material. Two obvious regional candidate genes, encoding muscular proteins became subjects for sequence analyses, the gene for myosin light chain 1 slow-twitch muscle A on 12q13 and the muscle specific exons of ankyrin 1 on 8p11. No mutations were identified in the coding sequence.