Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease

The Development of Linguistic Competences for Employability: A Training Project for Teachers

AbstractEmployability is a new concept that has just appeared in the Spanish educational system. Its rising importance is due to European Union educational policies which aim to provide young people with training that enables them to take part successfully in the present and future working world.This paper argues for the need to develop employability from the very start of formal education, and within this, we highlight the importance of developing linguistic competence among pre-school and primary pupils as a key element for favouring employability.To be able to do so, the teaching staff must be trained using quality education to enable them to work effectively on this competence. In this paper we present how a training program, with a specific European dimension, has been designed by a state school from the Valencian Community, to serve as a model for other schools concerned about the development of a linguistic competence that helps to improve both teachers’ and pupils’ employability

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease

Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics

<div><p>During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN) progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.</p></div

Olig2 coordinates patterning and neuronal differentiation.

<p>(A) Proposed model of the Olig2-controlled gene regulatory network. Olig2 not only acts as central organizer for dorsal-ventral patterning in the spinal cord but also controls the rate of MN differentiation through direct repression of Hes TFs. This leads to a higher levels of Ngn2 expression and, consequently, a higher rate of neuronal differentiation in the pMN domain, compared to adjacent progenitor domains. (B) Olig2 is a core component of the Shh-controlled gene regulatory network that patterns the ventral spinal cord [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.ref006" target="_blank">6</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.ref067" target="_blank">67</a>]. (C) Olig2-mediated down-regulation of the Notch effectors Hes1/5 relieves repression of Ngn2 in the pMN domain. (D) Consolidated activities of Ngn2 and Olig2 cause differentiation of NPs to MNs. Olig2 promotes differentiation of MNs through repression of alternative IN cell fates. bHLH, basic helix-loop-helix; IN, interneuron; MN, motor neuron; NP, neural progenitor; pMN, MN progenitor; p2, V2 interneuron progenitor; p3, V3 interneuron progenitor; RA, retinoic acid; Shh, sonic hedgehog; TF, transcription factor; V2, V2 interneuron; V3, V3 interneuron.</p

Olig2 binds to an evolutionarily conserved element near Hes5.

<p>(A) Identification of an evolutionarily conserved element containing an E-box in the vicinity of the <i>Hes5</i> genomic locus in chick, mouse, and human (Hes5(e1)). (B) Analysis of Olig2 Chip-Seq data from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.ref063" target="_blank">63</a>] reveals Olig2 binding sites in the vicinity of the <i>Hes1</i> and <i>Hes5</i> genes. The peak corresponding to the Hes5(e1) element is highlighted in red. (C) Electrophoretic mobility shift assays show that both Olig2 and E12 homodimers can individually bind to the Hes5(e1) E-box and do not form any heterodimeric complexes (lanes 1–4). Positions of the different protein complexes are indicated by colored arrows. Binding depends on the E-box, as both proteins fail to bind probes containing an E-box mutation (Hes5(e1ΔE)) (lanes 5–7). Olig2 binding to Hes5(e1) can be abolished by the addition of unlabelled Hes5(e1) probes, but not those containing the E-box mutation (lanes 8–14). (D) Id1 inhibits binding of E12, but not of Olig2 or Ngn2, to the Hes5(e1) element. Olig2, E12, and Ngn2 alone or Ngn2/E12 heterodimers can bind the Hes5(e1) element. Mixing Olig2 or Ngn2 with Id1 does not inhibit their homodimeric binding activities (lanes 2, 5, 8, and 10). In contrast, Id1 strongly inhibits binding of both E12/E12 and Ngn2/E12 complexes (lanes 6 and 10). The addition of E12 without and with Id1 does not affect Olig2 binding efficiency (lanes 2, 4, and 7). ATG, translational initation codon; Chip-Seq, chromatin immunoprecipitation-sequence; E-box, bHLH transcription factor binding site; N2, Ngn2 protein; O2, Olig2 protein.</p

Characterization of MN differentiation from ESCs.

<p>(A) Scheme outlining the differentiation protocol. ESCs are plated in N2B27 + FGF for 2 days before being exposed to N2B27 + FGF/CHIR, resulting in the production of NMPs at day 3. Cells are subsequently exposed to RA and SAG to promote differentiation into ventral NPs and MNs. (B, C) Expression of NP (Pax6, Olig2, Nkx2.2, Sox1) and MN (Isl1/2) markers between day 4 and day 7 in differentiating ESCs. (D) RT-qPCR analysis of <i>Irx3</i>, <i>Pax6</i>, <i>Nkx6</i>.<i>1</i>, <i>Olig2</i>, and <i>Nkx2</i>.<i>2</i> expression from day 3 to day 7 reveals progressive ventralization in response to increasing duration of Shh signaling. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.s013" target="_blank">S1 Data</a>. (E) MN induction after day 5, revealed by RT-qPCR analysis of <i>Sox1</i>, <i>Ngn2</i>, <i>Isl1</i>, and <i>Tubb3</i>. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.s013" target="_blank">S1 Data</a>. Scale bars = 40 μm. CHIR, Wnt pathway activator CHIR99021; ESC, embryonic stem cell; FGF, fibroblast growth factor 2; MN, motor neuron; NMP, neuromesodermal progenitor; NP, neural progenitor; N2B27, N2 and B27 media supplements; RA, retinoic acid; RT-qPCR, real-time quantitative polymerase chain reaction; SAG, Smoothened/Shh signalling agonist.</p

The Hes5(e1) element is required for repression of reporter genes in the pMN domain.

<p>(A, B) Co-electroporation of CMV/β-actin::nLacZ and Hes5(e1) reporter plasmids into chick spinal cord. Although electroporation (revealed by β-Gal antibody staining, magenta in [A]) is uniform along the dorsal-ventral axis, expression of the EGFP reporter is confined to intermediate parts of the neural tube (A, B), and little coexpression of Olig2 and EGFP was detected (B). (C) Design of Hes5(e1) and Hes5(e1ΔE) reporters. The Hes5(e1) element was cloned in front of β-globin minimal promoter to drive <i>EGFP</i> reporter gene expression. To test the importance of the E-box in the Hes5(e1) element, critical base pairs for Olig2 binding were mutated (red). (D, E) Co-electroporation of CMV/β-actin::-nLacZ and Hes5(e1ΔE) reporter plasmids into chick spinal cord. In contrast to the Hes5(e1) reporter plasmid, significant coexpression of Olig2 and GFP in the pMN domain is detected (E). Note that E-box mutation reduced the basal activity of the reporter such that longer exposure times were needed to achieve the signal levels seen in the intermediate spinal cord with the nonmutated Hes5(e1) reporter (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.s008" target="_blank">S8B and S8C Fig</a>). (F) Scatter dot plots display the dorsal-ventral positions (distance from the roof plate) of individual cells expressing the Hes5(e1) and Hes5(e1ΔE) reporters, relative to CMV/β-actin::-nLacZ and Olig2. Results are aggregated from five representative sections taken from five well-electroporated and stage-matched spinal cords. The Hes5(e1ΔE) reporter exhibits a significant ventral shift in its activity and considerable overlap with Olig2 expression (blue dotted box). Box plots include the median and whiskers represent 5th and 95th percentiles. Data points that lay outside the DV scale used to assess these experiments were excluded from this analysis. ** <i>p</i> = 0.0005, Mann-Whitney test; <i>p</i> = 0.6649. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003127#pbio.2003127.s013" target="_blank">S1 Data</a>. (G) EGFP expression in Hes5(e1)-nEGFP whole mount embryos at e10.5. (H–H″) Cryosections of Hes5(e1)-nEGFP embryos at e10.5 assayed for GFP, Olig2, and Hes5. EGFP expression colocalizes with Hes5 expression (H″) but not with Olig2 (H). (I–N) Hes5(e1)-nEGFP expression in <i>Olig2</i> heterozygous (I, K, L) and homozygous mutants (J, M, N). In <i>Olig2</i> heterozygotes, little nEGFP expression can be detected in the Olig2 expression domain, resulting in a pronounced gap between the expression domains of EGFP, Nkx2.2, and Hes1 (K, L). By contrast, the EGFP, Nkx2.2, and Hes1 expression domains directly abut each other in <i>Olig2</i> homozygous mutants (M, N). β-Gal, beta-galactosidase; βGlob, beta-globin; CMV/β-actin::nLacZ, cytomegalovirus/chick beta-actin promoter driving nuclear LacZ gene expression; ΔE, E-box deletion; E-box, bHLH protein binding site; EGFP, enhanced green fluorescent protein; FP, floor plate; GFP, green fluorescent protein; H5(e1), Hes5(e1) genomic element; ns, not significant; pMN, motor neuron progenitor; WT, wild-type.</p

Olig2 and Hes are dynamically expressed in the mouse neural tube.

<p>(A–D) Expression patterns of Ngn2 (green in A), Olig2 (red in A, C, D), Hes1 (red in B, green in C), and Hes5 (green in B, D) in the neural tube at e10.5. Note the low expression levels of Hes1/5 and high expression levels of Ngn2 in the pMN domain (compare A, B). (E) Hes5 (green) expression coincides with the expression of high levels of Pax6 (red) in the intermediate neural tube. (F, G) Hes1 expression (green) is readily detected in both Nkx2.2⁺ p3 progenitors (red in F) and floor plate cells labelled by Foxa2 expression (red in G). (H–Q′) Time course of Olig2 (blue), Hes1 (red), Hes5 (red), and Ngn2 (green) expression in neural tubes between e8.5 and e10.5. Multiple panels shown for e9.5 reflect developmental progression from caudal to rostral positions along the neuraxis. Hes1 expression appears to recede from the ventral neural tube upon the onset of Olig2 expression at e8.5 (H) and is thereafter absent from most Olig2+ cells (I–L). Olig2 and Hes5 are initially coexpressed (M, N). Over time, Hes5 expression progressively disappears from the pMN domain (N–Q), and Ngn2 concomitantly increases (N′–Q′). Insets show single channel images of the outlined area for the respective markers. Scale bars = 50 μm. e, embryonic day; pMN, MN progenitor; p3, V3 interneuron progenitor.</p