Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals

Hox genes controlling motor neuron subtype identity are expressed in rostrocaudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains that are controlled by rostrocaudal patterning signals that trigger rapid, domain-wide clearance of repressive histone H3 Lys27 trimethylation (H3K27me3) polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid leads to activation and binding of retinoic acid receptors (RARs) to the Hox1–Hox5 chromatin domains, which is followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and fibroblast growth factor (FGF) signals induce expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1–Hox9 chromatin domains, leading to specification of brachial or thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostrocaudal neural identity and that maintenance of these chromatin domains ensures the transmission of positional identity to postmitotic motor neurons later in development.Leona M. and Harry B. Helmsley Charitable TrustNational Institutes of Health (U.S.) (Grant P01 NS055923)Smith Family Foundatio

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived cultures

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived culturesHuman motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.This work was funded by Project A.L.S., P2ALS and NYSTEM grant number CO24415. The work of N.J.L. was supported by the Portuguese Foundation for Science and Technology SFRH/BD/33421/2008 and the Luso-American Development Foundation. B.J.-K. was supported by the National Institute of Neurological Disorders and Stroke (NINDS). L.R. was supported by the Swedish Brain Foundation/Hjarnfonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.National Institutes of Health (U.S.) (Grant R01 HG005287

Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.Project ALS FoundationNational Institutes of Health (U.S.) (Grant P01 NS055923

Occupancy maps of 208 chromatin-associated proteins in one human cell type

Transcription factors are DNA-binding proteins that have key roles in gene regulation. Genome-wide occupancy maps of transcriptional regulators are important for understanding gene regulation and its effects on diverse biological processes. However, only a minority of the more than 1,600 transcription factors encoded in the human genome has been assayed. Here we present, as part of the ENCODE (Encyclopedia of DNA Elements) project, data and analyses from chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–seq) experiments using the human HepG2 cell line for 208 chromatin-associated proteins (CAPs). These comprise 171 transcription factors and 37 transcriptional cofactors and chromatin regulator proteins, and represent nearly one-quarter of CAPs expressed in HepG2 cells. The binding profiles of these CAPs form major groups associated predominantly with promoters or enhancers, or with both. We confirm and expand the current catalogue of DNA sequence motifs for transcription factors, and describe motifs that correspond to other transcription factors that are co-enriched with the primary ChIP target. For example, FOX family motifs are enriched in ChIP–seq peaks of 37 other CAPs. We show that motif content and occupancy patterns can distinguish between promoters and enhancers. This catalogue reveals high-occupancy target regions at which many CAPs associate, although each contains motifs for only a minority of the numerous associated transcription factors. These analyses provide a more complete overview of the gene regulatory networks that define this cell type, and demonstrate the usefulness of the large-scale production efforts of the ENCODE Consortium

In vivo assembly of the axon initial segment in motor neurons

International audienceThe axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and beta 4-spectrin only appear along AnkG(+) segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither beta 4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG

How to Build Transcriptional Network Models of Mammalian Pattern Formation

Genetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations. allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20–30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain.nalysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning

Hoxb1 Controls Anteroposterior Identity of Vestibular Projection Neurons

The vestibular nuclear complex (VNC) consists of a collection of sensory relay nuclei that integrates and relays information essential for coordination of eye movements, balance, and posture. Spanning the majority of the hindbrain alar plate, the rhombomere (r) origin and projection pattern of the VNC have been characterized in descriptive works using neuroanatomical tracing. However, neither the molecular identity nor developmental regulation of individual nucleus of the VNC has been determined. To begin to address this issue, we found that Hoxb1 is required for the anterior-posterior (AP) identity of precursors that contribute to the lateral vestibular nucleus (LVN). Using a gene-targeted Hoxb1-GFP reporter in the mouse, we show that the LVN precursors originate exclusively from r4 and project to the spinal cord in the stereotypic pattern of the lateral vestibulospinal tract that provides input into spinal motoneurons driving extensor muscles of the limb. The r4-derived LVN precursors express the transcription factors Phox2a and Lbx1, and the glutamatergic marker Vglut2, which together defines them as dB2 neurons. Loss of Hoxb1 function does not alter the glutamatergic phenotype of dB2 neurons, but alters their stereotyped spinal cord projection. Moreover, at the expense of Phox2a, the glutamatergic determinants Lmx1b and Tlx3 were ectopically expressed by dB2 neurons. Our study suggests that the Hox genes determine the AP identity and diversity of vestibular precursors, including their output target, by coordinating the expression of neurotransmitter determinant and target selection properties along the AP axis

Functional Neuromuscular Junctions Formed by Embryonic Stem Cell-Derived Motor Neurons

A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN) disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease

Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state

A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro