Ulrika Marklund

Lmx1b is essential for the development of serotonergic neurons

The specification and differentiation of serotonergic (5-HT) neurons require both extrinsic signaling molecules and intrinsic transcription factors to work in concert or in cascade. Here we identify the genetic cascades that control the specification and differentiation of 5-HT neurons in mice. A major determinant in the cascades is an LIM homeodomain-containing gene, Lmx1b, which is required for the development of all 5-HT neurons in the central nervous system. Our results suggest that, during development of 5-HT neurons, Lmx1b is a critical intermediate factor that couples Nkx2-2–mediated early specification with Pet1-mediated terminal differentiation. Moreover, our data indicate that genetic cascades controlling the caudal and rostral 5-HT neurons are distinct, despite their shared components.

Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%–95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.

Parasympathetic neurons originate from nerve-associated peripheral glial progenitors

Exploiting nervous paths already traveled The parasympathetic nervous system helps regulate the functions of many tissues and organs, including the salivary glands and the esophagus. To do so, it needs to reach throughout the body, connecting central systems to peripheral ones. Dyachuk et al. and Espinosa-Medina et al. explored how these connections are established in mice (see the Perspective by Kalcheim and Rohrer). Progenitor cells that travel along with the developing nerves can give rise to both myelinforming Schwann cells and to parasympathetic neurons. That means the interacting nerves do not have to find each other. Instead, the beginnings of the connections are laid down as the nervous system develops. Science, this issue p. 82, p. 87; see also p. 32 Parasympathetic neurons are born from Schwann cell precursors located in the nerves that carry preganglionic fibers. [Also see Perspective by Kalcheim and Rohrer] The peripheral autonomic nervous system reaches far throughout the body and includes neurons of diverse functions, such as sympathetic and parasympathetic. We show that the parasympathetic system in mice—including trunk ganglia and the cranial ciliary, pterygopalatine, lingual, submandibular, and otic ganglia—arise from glial cells in nerves, not neural crest cells. The parasympathetic fate is induced in nerve-associated Schwann cell precursors at distal peripheral sites. We used multicolor Cre-reporter lineage tracing to show that most of these neurons arise from bi-potent progenitors that generate both glia and neurons. This nerve origin places cellular elements for generating parasympathetic neurons in diverse tissues and organs, which may enable wiring of the developing parasympathetic nervous system.

A homeodomain feedback circuit underlies step-function interpretation of a Shh morphogen gradient during ventral neural patterning

The deployment of morphogen gradients is a core strategy to establish cell diversity in developing tissues, but little is known about how small differences in the concentration of extracellular signals are translated into robust patterning output in responding cells. We have examined the activity of homeodomain proteins, which are presumed to operate downstream of graded Shh signaling in neural patterning, and describe a feedback circuit between the Shh pathway and homeodomain transcription factors that establishes non-graded regulation of Shh signaling activity. Nkx2 proteins intrinsically strengthen Shh responses in a feed-forward amplification and are required for ventral floor plate and p3 progenitor fates. Conversely, Pax6 has an opposing function to antagonize Shh signaling, which provides intrinsic resistance to Shh responses and is important to constrain the inductive capacity of the Shh gradient over time. Our data further suggest that patterning of floor plate cells and p3 progenitors is gated by a temporal switch in neuronal potential, rather than by different Shh concentrations. These data establish that dynamic, non-graded changes in responding cells are essential for Shh morphogen interpretation, and provide a rationale to explain mechanistically the phenomenon of cellular memory of morphogen exposure.

Control of Notch-ligand endocytosis by ligand-receptor interaction

In Notch signaling, cell-bound ligands activate Notch receptors on juxtaposed cells, but the relationship between ligand endocytosis, ubiquitylation and ligand-receptor interaction remains poorly understood. To study the specific role of ligand-receptor interaction, we identified a missense mutant of the Notch ligand Jagged1 (Nodder, Ndr) that failed to interact with Notch receptors, but retained a cellular distribution that was similar to wild-type Jagged1 (Jagged1WT) in the absence of active Notch signaling. Both Jagged1WT and Jagged1Ndr interacted with the E3 ubiquitin ligase Mind bomb, but only Jagged1WT showed enhanced ubiquitylation after co-culture with cells expressing Notch receptor. Cells expressing Jagged1WT, but not Jagged1Ndr, trans-endocytosed the Notch extracellular domain (NECD) into the ligand-expressing cell, and NECD colocalized with Jagged1WT in early endosomes, multivesicular bodies and lysosomes, suggesting that NECD is routed through the endocytic degradation pathway. When coexpressed in the same cell, Jagged1Ndr did not exert a dominant-negative effect over Jagged1WT in terms of receptor activation. Finally, in Jag1Ndr/Ndr mice, the ligand was largely accumulated at the cell surface, indicating that engagement of the Notch receptor is important for ligand internalization in vivo. In conclusion, the interaction-dead Jagged1Ndr ligand provides new insights into the specific role of receptor-ligand interaction in the intracellular trafficking of Notch ligands.

Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression.

Homeodomain (HD) transcription factors and components of the Notch pathway [Delta1 (Dll1), Jagged1 (Jag1) and the Fringe (Fng) proteins] are expressed in distinct progenitor domains along the dorsoventral (DV) axis of the developing spinal cord. However, the internal relationship between these two regulatory pathways has not been established. In this report we show that HD proteins act upstream of Notch signalling. Thus, HD proteins control the spatial distribution of Notch ligands and Fng proteins, whereas perturbation of the Notch pathway does not affect the regional expression of HD proteins. Loss of Dll1 or Jag1 leads to a domain-specific increase of neuronal differentiation but does not affect the establishment of progenitor domain boundaries. Moreover, gain-of-function experiments indicate that the ability of Dll1 and Jag1 to activate Notch is limited to progenitors endogenously expressing the respective ligand. Fng proteins enhance Dll1-activated Notch signalling and block Notch activation mediated by Jag1. This finding, combined with the overlapping expression of Fng with Dll1 but not with Jag1, is likely to explain the domain-specific activity of the Notch ligands. This outcome is opposite to the local regulation of Notch activity in most other systems, including the Drosophila wing, where Fng co-localizes with Jagged/Serrate rather than Dll/Delta, which facilitates Notch signalling at regional boundaries instead of within domains. The regulation of Notch activation in the spinal cord therefore appears to endow specific progenitor populations with a domain-wide autonomy in the control of neurogenesis and prevents any inadequate activation of Notch across progenitor domain boundaries.

Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla

Following the yellow brick road The adrenal glands affect a variety of processes such as stress responses and metabolism. The mature adrenal gland is formed from multiple tissue sources, including cells of neural origin. Furlan et al. traced the origins of these cells. The cells first become Schwann cell precursors and follow along nerves to travel from the dorsal root ganglia of the spine to the adrenal gland. Once there, the cells differentiate into chromaffin cells. The authors used singlecell transcriptomics to reveal the shifts in functional programs during migration, development, and differentiation. Science, this issue p. eaal3753 The adrenal gland is built from cells that travel along highways of nerves. INTRODUCTION Circulating adrenaline can have profound effects on the body’s “inner world,” adjusting levels depending on demand to maintain organ and bodily homeostasis during daily living. In the more extreme fight-or-flight response, the surge of adrenaline is “energizing” through effects on organs and tissues, including increased heart rate and blood glucose levels, and redirecting oxygen and glucose to limb muscles. Chromaffin cells located in the adrenal medulla constitute the main hormonal component of the autonomic nervous system and are the principal source for release of catecholamines, including adrenaline, in the systemic circulation. Understanding the cellular origin and biological processes by which the adrenal medulla is formed during development is needed for mechanistic insights into how the hormonal component of the autonomic nervous system is formed and its relation to the rest of the autonomic nervous system. RATIONALE Adrenergic chromaffin cells in the adrenal medulla are thought to originate from a common sympathoadrenal lineage close to the dorsal aorta, where these cells split in a dorsoventral direction, forming the sympathetic chain and adrenal medulla, respectively. Revisiting this dogma, we examined the cell type origin of chromaffin cells, lineage segregation of sympathoblasts and chromaffin cells, the gene programs driving specification of chromaffin cells from progenitors, and the proliferative dynamics by which the adrenal medulla is formed. RESULTS We found that chromaffin cells of the adrenal medulla are formed from peripheral glia stem cells, termed Schwann cell precursors. Genetic cell lineage tracing revealed that most chromaffin cells arise from Schwann cell precursors, and consistently, genetic ablation of Schwann cell precursors results in marked depletion of chromaffin cells. Genetic ablation of the preganglionic nerve, on which Schwann cell precursors migrate, similarly leads to marked deficiencies of chromaffin cells, and fate-tracing cells unable to differentiate into chromaffin cells reveal an accumulation of glia cells in the region of the adrenal medulla. Experiments reveal that sympathetic and adrenergic lineages diverge at an unexpectedly early stage during embryonic development. Embryonic development of the adrenal medulla relies on recruitment of numerous Schwann cell precursors with limited cell expansion. Thus, the large majority of chromaffin cells arise from Schwann cell precursors migrating on preganglionic nerves innervating the adrenal medulla. Unexpectedly, single-cell RNA sequencing revealed a complex gene-regulatory mechanism during differentiation of Schwann cell precursors to chromaffin cells, whereby Schwann cell precursors enter into a gene expression program unique for a transient cellular state. Subsequently, this gene program and chromaffin cell gene networks suppress glial gene programs, advancing cells into the chromaffin cell identity. CONCLUSION By revisiting development of the adrenergic sympathetic system, we discovered a new cellular origin of this nervous system component. The adrenergic medulla is built from both neural crest cells and Schwann cell precursors, with a major contribution from Schwann cell precursors in rodents. A cellular origin from Schwann cell precursors highlights the importance of peripheral nerves as a stem cell niche and transportation routes for progenitors essential for neuroendocrine development. These results and mechanisms of differentiation through a transient intermediate cell type may also be helpful in advancing our knowledge on neuroblastoma and pheochromocytoma, because these most often arise from the adrenal gland region. Adrenal medulla largely originates from Schwann cell precursors. Overview of adrenal medulla development resulting from lineage tracing and nerve ablation experiments. SCP, Schwann cell precursor; AG, adrenal gland; NT, neural tube; n, notochord; DRG, dorsal root ganglion; IML, intermediolateral column; NCC, neural crest cells; NC, neural crest; DA, dorsal aorta; SRG, suprarenal sympathetic ganglion. Red encodes early NCCs and their derivatives. Blue encodes late neural crest and SCP-derived cell types. Adrenaline is a fundamental circulating hormone for bodily responses to internal and external stressors. Chromaffin cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland, where they detach from the nerve and form postsynaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell type specification. Subsequently, these programs down-regulate SCP-gene and up-regulate chromaffin cell–gene networks. The AM forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development.

Detailed Expression Analysis of Regulatory Genes in the Early Developing Human Neural Tube

Studies in model organisms constitute the basis of our understanding of the principal molecular mechanisms of cell fate determination in the developing central nervous system. Considering the emergent applications in stem cell-based regenerative medicine, it is important to demonstrate conservation of subtype specific gene expression programs in human as compared to model vertebrates. We have examined the expression patterns of key regulatory genes in neural progenitor cells and their neuronal and glial descendants in the developing human spinal cord, hindbrain, and midbrain, and compared these with developing mouse and chicken embryos. As anticipated, gene expression patterns are highly conserved between these vertebrate species, but there are also features that appear unique to human development. In particular, we find that neither tyrosine hydroxylase nor Nurr1 are specific markers for mesencephalic dopamine neurons, as these genes also are expressed in other neuronal subtypes in the human ventral midbrain and in human embryonic stem cell cultures directed to differentiate towards a ventral mesencephalic identity. Moreover, somatic motor neurons in the ventral spinal cord appear to be produced by two molecularly distinct ventral progenitor populations in the human, raising the possibility that the acquisition of unique ventral progenitor identities may have contributed to the emergence of neural subtypes in higher vertebrates.

Molecular Architecture of the Mouse Nervous System

Summary The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.

Ascl1 Is Required for the Development of Specific Neuronal Subtypes in the Enteric Nervous System.

The enteric nervous system (ENS) is organized into neural circuits within the gastrointestinal wall where it controls the peristaltic movements, secretion, and blood flow. Although proper gut function relies on the complex neuronal composition of the ENS, little is known about the transcriptional networks that regulate the diversification into different classes of enteric neurons and glia during development. Here we redefine the role of Ascl1 (Mash1), one of the few regulatory transcription factors described during ENS development. We show that enteric glia and all enteric neuronal subtypes appear to be derived from Ascl1-expressing progenitor cells. In the gut of Ascl1−/− mutant mice, neurogenesis is delayed and reduced, and posterior gliogenesis impaired. The ratio of neurons expressing Calbindin, TH, and VIP is selectively decreased while, for instance, 5-HT+ neurons, which previously were believed to be Ascl1-dependent, are formed in normal numbers. Essentially the same differentiation defects are observed in Ascl1KINgn2 transgenic mutants, where the proneural activity of Ngn2 replaces Ascl1, demonstrating that Ascl1 is required for the acquisition of specific enteric neuronal subtype features independent of its role in neurogenesis. In this study, we provide novel insights into the expression and function of Ascl1 in the differentiation process of specific neuronal subtypes during ENS development. SIGNIFICANCE STATEMENT The molecular mechanisms underlying the generation of different neuronal subtypes during development of the enteric nervous system are poorly understood despite its pivotal function in gut motility and involvement in gastrointestinal pathology. This report identifies novel roles for the transcription factor Ascl1 in enteric gliogenesis and neurogenesis. Moreover, independent of its proneurogenic activity, Ascl1 is required for the normal expression of specific enteric neuronal subtype characteristics. Distinct enteric neuronal subtypes are formed in a temporally defined order, and we observe that the early-born 5-HT+ neurons are generated in Ascl1−/− mutants, despite the delayed neurogenesis. Enteric nervous system progenitor cells may therefore possess strong intrinsic control over their specification at the initial waves of neurogenesis.