Understanding axial progenitor biology in vivo and in vitro

The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the in vitro production of axial progenitors and their potential implications in disease modelling and regenerative medicine

A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation

LISBOA-01?0145-FEDER-030254 SCML-MC-60-2014 LISBOA-01-0145-FEDER-022170 PD/BD/128426/2017 PD/BD/128437/2017 MR/S008799/1 MR/ K011200/1 DEV-170806Formation of the vertebrate postcranial body axis follows two sequential but distinct phases. The first phase generates pre-sacral structures (the so-called primary body) through the activity of the primitive streak on axial progenitors within the epiblast. The embryo then switches to generate the secondary body (post-sacral structures), which depends on axial progenitors in the tail bud. Here we show that the mammalian tail bud is generated through an independent functional developmental module, concurrent but functionally different from that generating the primary body. This module is triggered by convergent Tgfbr1 and Snai1 activities that promote an incomplete epithelial to mesenchymal transition on a subset of epiblast axial progenitors. This EMT is functionally different from that coordinated by the primitive streak, as it does not lead to mesodermal differentiation but brings axial progenitors into a transitory state, keeping their progenitor activity to drive further axial body extension.publishersversionpublishe

In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity

Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm in vitro and in vivo. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types in vitro and the analysis of previous difficult to access aspects of embryo development

A gene regulatory network balances neural and mesoderm specification during vertebrate trunk development

Transcriptional networks, regulated by extracellular signals, control cell fate decisions and determine the size and composition of developing tissues. One example is the network controlling bipotent neuromesodermal progenitors (NMPs) that fuel embryo elongation by generating spinal cord and trunk mesoderm tissue. Here, we use single-cell transcriptomics to identify the molecular signature of NMPs and reverse engineer the mechanism that regulates their differentiation. Together with genetic perturbations, this reveals a transcriptional network that integrates opposing retinoic acid (RA) and Wnt signals to determine the rate at which cells enter and exit the NMP state. RA, produced by newly generated mesodermal cells, provides feedback that initiates NMP generation and induces neural differentiation, thereby coordinating the production of neural and mesodermal tissue. Together, the data define a regulatory network architecture that balances the generation of different cell types from bipotential progenitors in order to facilitate orderly axis elongation

Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Background: Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods: We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results: Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions: Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency

Generation and characterisation of hNMPs.

<p>(A) Scheme describing the culture conditions employed for neural differentiation of hES cells treated for 72 h either with FGF/CHIR or subjected to dual SMAD inhibition (LDN, LDN193189; SB43, SB431542). (B) BRACHYURY/SOX2 immunocytochemistry in undifferentiated and FGF/CHIR-treated (48 h) hES cells. Corresponding graphs depict image analysis of BRACHYURY and SOX2 expression in the indicated culture conditions. Numbers: percentages of cells in each quadrant. (C) qPCR analysis for indicated markers in hES cells treated with FGF/CHIR for 72 h (D3) or 96 h (D4). Error bars = s.d. (n = 2). Results are represented as log₁₀ ratio of expression versus untreated hES cells. (D) qPCR analysis for indicated differentiation markers in hES cells differentiated in N2B27 following either an NM progenitor induction- (N_P) or a dual SMAD inhibition-intermediate step (N_A). Error bars = s.d. (n = 2). Anterior, anterior neural markers; PXM, paraxial mesoderm; n/d, not determined. (E) Immunocytochemistry for SOX2/HOXC8 in N_A and N_P culture conditions indicated in the scheme (A). (F) Quantitation of the coexpression of Hoxc8 with Sox2 in N_A and N_P conditions. All data used to generate the plots can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s013" target="_blank">Data S5</a>.</p

Generation of NMPs from EpiSCs.

<p>(A) Brachyury/Sox2 immunocytochemistry in EpiSC cultures treated with FGF/CHIR for 72 h. (B) qPCR analysis for indicated markers in mouse EpiSCs treated with FGF/CHIR. Error bars = s.d. (n = 3). n/d, not determined. Results are represented as log₁₀ ratio of expression versus untreated EpiSCs. The data used to generate the plot can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s012" target="_blank">Data S4</a>. (C) Combined fluorescence/brightfield microscopy showing donor cell incorporation of grafted GFP⁺ EpiSC differentiated for 48 h in FGF/CHIR after 48 h embryo culture. (D) Table summarizing the incorporation of grafted GFP⁺ EpiSC differentiated for 24 h or 48 h in Fgf/Wnt within host embryos. NT, neural tube; Som, somite; PSM, presomitic mesoderm; n/a, not applicable. (E) Representative examples of donor cell incorporation (green, GFP) and differentiation (red, immunofluorescence for indicated markers). Cell nuclei were stained with DAPI (blue). White boxes indicate the position of magnified images of GFP⁺ cells.</p

Transient Wnt and FGF signalling induce dual fated neuromesodermal progenitors.

<p>(A) Schematic of differentiation protocols used to generate mesoderm and neural cells from a common NM progenitor population. (B) mRNA-seq expression values of <i>Sox2</i>, <i>Brachyury</i>, <i>Tbx6</i> and <i>Cdx2</i> following exposure to bFGF alone or bFGF/CHIR for 12 h (D2.5) and 24 h (D3). Activation of Wnt signalling with CHIR upregulated Brachyury within 12 h. Expression of <i>Tbx6</i> and <i>Cdx2</i> was also upregulated in NMPs by D3, whereas <i>Sox2</i> transcript levels were decreased. (C) Immunostaining of cells treated with FGF/Wnt revealed the coexpression of Brachyury with Sox2 (NMPs). In the absence of Wnt, NPCs express Sox2 but the expression of Brachyury is only evident in a very small proportion of cells. (D) mRNA expression values of neural (<i>Sox1</i>, <i>Sox2</i>, <i>Sox3</i>) and mesodermal progenitors markers (<i>Tbx6</i>, <i>Bra</i>, <i>Msgn1</i>) in posterior neural (N_P) and mesodermal cells (Meso) at D5 show the generation of distinct populations depending on treatment after D3. Removal of Wnt at D3 results in the generation of neural cells expressing Sox1–3 whereas continued Wnt exposure induces expression of Tbx6, Brachyury and Msgn1, characteristic of paraxial mesodermal. (E) Immunostaining indicates that continued Wnt exposure generates paraxial mesodermal progenitors that express Tbx6 at D5 and Desmin and MyoD at D8. (F) Sketch of a chick embryo (HH8–9) showing the injection site (IS) of NMP or N_A cells. (G) NMP cells were labelled with DiI and transplanted in the CLE region. After 24 h the cells had incorporated into both the neural tube and somites. Whole-mount and transverse sections of HH17 chick embryos show the incorporation (asterisks) in the neural tube (H) and somites (I). (J) Table summarizing the number of chick embryos that were injected at stage HH8–9 and had engrafted cells in the neural tube, the somites or both 24 h later. Injection of N_A cells resulted in incorporation only in the neural tube (K, L). All data used to generate the plots of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g002" target="_blank">Figure 2</a> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s010" target="_blank">Data S2</a>.</p

Generation of neural cells with specific AP identities from ESCs.

<p>(A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N_A), Hindbrain (N_H) and Spinal cord (N_P) identities. (B) Relative expression levels of the indicated genes from N_A, N_H and N_P cells at day 5 (D5) of differentiation indicate that N_A, N_H and N_P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N_A cells express high levels of forebrain markers including <i>Otx1</i> and <i>Otx2</i>; N_H cells express genes characteristic of hindbrain including <i>Mafb</i> and <i>Hoxa2</i> genes; N_P cells express high levels of posterior 5′ Hox genes including <i>Hoxc8</i> and <i>Hoxc9</i>. The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N_H and N_P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N_P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937-Kmita1" target="_blank">[74]</a>. In N_H cells <i>Hoxb1</i> and <i>Hoxb2</i> are induced prior to <i>Hoxc4</i>. However, the more posterior Hox genes are not induced. By contrast, in N_P conditions the 5′ Hox genes <i>Hoxc6</i>, <i>Hoxc8</i> and <i>Hoxc9</i> are induced at D4 and their expression is maintained at day 5. (Note log₂ scale). (D) Immunohistochemistry indicates that N_H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N_P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N_H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N_P conditions. All data used to generate the plots of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio-1001937-g001" target="_blank">Figure 1</a> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s009" target="_blank">Data S1</a>.</p

Brachyury is necessary for mesoderm formation but not posterior neural identity.

<p>(A) Schematic of the conditions used for mesoderm differentiation. (B) qRT-PCR analysis of the expression of <i>Tbx6</i>, <i>Cdx2</i> and <i>Hoxb1</i> relative to b-actin at D3 of differentiation in wild-type (wt) and Brachyury null cells (Bra^−/−) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while <i>Cdx2</i> and <i>Hoxb1</i> continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra^−/− ESCs exposed to CHIR from D2–D5 (Meso conditions). Posterior Hox genes <i>Hoxc8</i> and <i>Hoxc9</i> are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers <i>Sox1</i> and <i>Sox2</i> are expressed only in Bra^−/− cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra^−/− and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra^−/− cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not β-Tubulin (Tuj1) whereas Bra^−/− cells fail to produce Desmin/MyoD and differentiate into neurons expressing β-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N_P) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log₂₊ scale). All data used for the plots can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001937#pbio.1001937.s014" target="_blank">Data S6</a>. (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling.</p