Lia Panman

Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling

Spatially and temporally coordinated changes in gene expression are crucial to orderly progression of embryogenesis. We combine mouse genetics with experimental manipulation of signalling to analyze the kinetics by which the SHH morphogen and the BMP antagonist gremlin 1 (GREM1) control gene expression in the digit-forming mesenchyme of mouse limb buds. Although most mesenchymal cells respond rapidly to SHH signalling, the transcriptional upregulation of specific SHH target signals in the mesenchyme occurs with differential temporal kinetics and in a spatially restricted fashion. In particular, the expression of the BMP antagonist Grem1 is always upregulated in mesenchymal cells located distal to the SHH source and acts upstream of FGF signalling by the apical ectodermal ridge. GREM1/FGF-mediated feedback signalling is, in turn, required to propagate SHH and establish the presumptive digit expression domains of the Notch ligand jagged 1 (Jag1) and 5′Hoxd genes in the distal limb bud mesenchyme. Their establishment is significantly delayed in Grem1-deficient limb buds and cannot be rescued by specific restoration of SHH signalling in mutant limb buds. This shows that GREM1/FGF feedback signalling is required for regulation of the temporal kinetics of the mesenchymal response to SHH signalling. Finally, inhibition of SHH signal transduction at distinct time points reveals the differential temporal dependence of Grem1, Jag1 and 5′Hoxd gene expression on SHH signalling. In particular, the expression of Hoxd13 depends on SHH signal transduction significantly longer than does Hoxd11 expression, revealing that the reverse co-linear establishment, but not maintenance of their presumptive digit expression domains, depends on SHH signalling.

NR4A orphan nuclear receptors as mediators of CREB-dependent neuroprotection

Induced expression of neuroprotective genes is essential for maintaining neuronal integrity after stressful insults to the brain. Here we show that NR4A nuclear orphan receptors are induced after excitotoxic and oxidative stress in neurons, up-regulate neuroprotective genes, and increase neuronal survival. Moreover, we show that NR4A proteins are induced by cAMP response element binding protein (CREB) in neurons exposed to stressful insults and that they function as mediators of CREB-induced neuronal survival. Animals with null mutations in three of six NR4A alleles show increased oxidative damage, blunted induction of neuroprotective genes, and increased vulnerability in the hippocampus after treatment with kainic acid. We also demonstrate that NR4A and the transcriptional coactivator PGC-1α independently regulate distinct CREB-dependent neuroprotective gene programs. These data identify NR4A nuclear orphan receptors as essential mediators of neuroprotection after exposure to neuropathological stress.

Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development

The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.

Transcription Factor-Induced Lineage Selection of Stem-Cell-Derived Neural Progenitor Cells

The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogeneous neuronal cultures. Here we show that transcription factors like Lmx1a, Phox2b, Nkx2.2, and Olig2 can induce desired neuronal lineages from most expressing neural progenitor cells by a mechanism resembling developmental binary cell-fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene-expression validation of generated neurons and identification of previously unrecognized features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into specific neuronal cell types; e.g., dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and also promote efficient reprogramming in more remote neural contexts, including human neural progenitor cells.

Sox6 and Otx2 Control the Specification of Substantia Nigra and Ventral Tegmental Area Dopamine Neurons

Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinsons disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinsons disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.

Transcription Factor‐Induced Lineage Programming of Noradrenaline and Motor Neurons from Embryonic Stem Cells

An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell‐derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid‐ and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome‐wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type. Stem Cells 2014;32:609–622

Tracing lineages to uncover neuronal identity

Many previous studies have focused on understanding how midbrain dopamine neurons, which are implicated in many neurological conditions, are generated during embryogenesis. One of the remaining questions concerns how different dopamine neuron subtypes are specified. A recent paper in Neural Development has revealed features of a spatial and temporal lineage map that, together with other studies, begins to elucidate the developmental origin of distinct neuronal subtypes within the developing midbrain.See research article http://www.neuraldevelopment.com/content/6/1/29

The Parkinson’s Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration

Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson’s disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an “upstream” oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-d-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis.

14-P020 Intrinsic transcriptional determinants promote efficient generation of neuronal subtypes from ES cells

Netrin1 is a member of a family of secreted molecules implicated in axon guidance, neuronal migration and apoptosis during development of central nervous system (CNS). Netrin1 signals in CNS through a group of transmembrane receptors belonging to the DCC (deleted in colorectal cancer) and C. elegans Unc5-related families. Netrin1 mutant mice die usually at birth with several CNS defects, but heterozygous mice are normal. We made Netrin1 expression analysis in developing and postnatal mouse brain. Netrin1 was strongly expressed in the proliferative ventricular zone (VZ) in embryonic brain and in the developmentally related ependymal layer of the postnatal brain. Isolated Netrin1 positive neurospheres from VZ/ependymal layer expressed several neural stem cell markers, were able to self-renew and differentiated into neurons, astrocytes and oligodendrocytes. Netrin1 expressing cells were also apparent in the rostral migratory stream (RMS), along which newly born neuroblasts migrate from lateral ventricles to the olfactory bulb contributing a constant renewal of the peripheral olfactory system. Netrin1 –/– mice had a smaller olfactory bulbs and immunohistological analysis revealed that Netrin1 expressing cells form a heterogenous subset of precursor cells contributing both neural and glial populations in olfactory bulb.