Gene expression in microdissected embryonic regions during mouse axis elongation.
Ontology highlight
ABSTRACT: This dataset contains spatiotemporal transcriptome information on different axial progenitors throughout mouse axis elongation. Neuromesodermal Progenitors (NMPs), Lateral and Paraxial Mesoderm Progenitors (LPMPs), and Notochord Progenitors (NotoPs) show distinct expression profiles. Extensive similarity exist between NMPs and their immediate mesoderm-committed descendants at each stage investigated. Over time transcriptional changes occur in LPMPs and NMPs, with the major change occurring in NMPs between early somitogenesis and completion of trunk morphogenesis. In contrast, NotoPs contain a more stable transcriptome over time.
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). NMPs reside in the caudal lateral epiblast and tailbud, from where they sustain axial elongation. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm, single-cell RNA sequencing (scRNA-seq) was performed on cells isolated from the tailbuds of control, Pbx1/Pbx2 compound (Pbx1/2-com) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) embryos at embryonic days 8.5 and 9.0. Single cells from at least three embryos per genotype and stage were FACS-sorted into 384-well capture plates, and scRNA-seq was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). As a spike-in internal control for batch-effect correction, 71 EpiSCs cultivated in vitro were sorted into each plate, together with 311 cells from embryonic tailbuds. Two wells were left empty in each plate, as a no-cell control during data analysis.
Project description:In the past decades, the paradigm of three germ layers formed by gastrulation has been modified by data suggesting an existence of neuromesodermal progenitors (NMPs) that arise during gastrulation and contribute to both spinal cord and adjacent paraxial mesoderm1-5. However, there lacks direct genetic lineage tracing evidence and functional assessment of NMPs in vivo. Here, we develop a dual recombinases-mediated genetic system to specifically trace and genetically ablate Brachyury+Sox2+ NMPs. Genetic lineage tracing results and single-cell RNA sequencing analysis show that NMPs contain three distinct uni-potent and bi-potent progenitor populations for progressive differentiation into neural and mesodermal fates. Genetic ablation of NMPs by diphtheria toxin reveals a critical role of NMPs in tail formation. This study provides in vivo genetic evidence for heterogeneity of NMPs in their cell fate determination and their functional role in developing embryos.
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm (PSM), wild-type (WT) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) EpiSCs were differentiated in vitro to PSM. Single cells from different time-points (WT: EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours; Pbx1/2-DKO: 24 hours, 36 hours, 48 hours) were FACS-sorted into 384-well capture plates, and single-cell RNA-sequencing was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). Two wells were left empty in each plate, as a no-cell control during data analysis.
Project description:The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4- dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX proteins play a fundamental role in promoting the expression of PM genes, including the master regulator Mesogenin1 (Msgn1). To address the role of PBX proteins in PM differentiation, RNA-seq was performed on wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (12 hours and 24 hours). To establish the direct transcriptional regulation of Msgn1 by PBX/HOX, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed RNA-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at 24 hours. All differentiation experiments were performed in biological triplicates with WT and Pbx1/2-DKO lines, and in biological duplicates with pMsgn1-mut lines.
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). WNT signalling sustains both NMP expansion and PM differentiation, but the mechanism by which it distinguishes between these alternative fates is unknown. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX proteins generate the DNA-binding context that allows recruitment of the WNT-effector LEF1, and consequently promote the expression of PM genes. Here, we wanted to identify the chromatin binding pattern of PBX1, PBX2 and LEF1 during the first 48 hours of pre-somitic mesoderm (PSM) in vitro differentiation, by performing chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) at different time-points (EpiSCs, 6 hours, 12 hours, 24 hours, 48 hours). To address the impact of PBX loss on LEF1 recruitment to chromatin, we additionally performed ChIP-seq in wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to PSM at different time-points (EpiSCs, 12 hours, 24 hours, 48 hours).
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). WNT signalling sustains both NMP expansion and PM differentiation, but the mechanism by which it distinguishes between these alternative fates is unknown. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX/HOX complexes establish a permissive chromatin landscape for de novo recruitment of the WNT-effector LEF1 on PM genes, including the master regulator Mesogenin1 (Msgn1). To assess the PBX-dependent changes in chromatin accessibility during PM differentiation, we performed ATAC-seq of wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours). To assess the direct consequence of PBX/HOX binding on chromatin accessibility, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed ATAC-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at different time-points (12 hours, 24 hours, 36 hours, 48 hours).
Project description:Progress has been made in generating spinal cord and trunk derivatives from neuromesodermal progenitors (NMPs). However, maintaining the self-renewal of NMPs in vitro remains a challenge. In this study, we developed a cocktail of small molecules and growth factors that induces human embryonic stem cells to produce self-renewing NMPs (srNMPs) under chemically defined conditions. These srNMPs maintain the state of thoracic neuromesodermal progenitors in prolonged culture and have the bipotential to generate mesodermal cells and neurons, even at the single-cell level. Additionally, suspended srNMP aggregates could partially mimic the structural properties of early embryonic trunks. Furthermore, transplanted srNMP-derived muscle satellite cells or progenitors of motor neurons were integrated into skeletal muscle or the spinal cord, respectively, and contributed to regeneration in mouse models. In summary, srNMPs hold great promise for applications in developmental biology and as renewable cell sources for cell therapy for trunk and spinal cord injuries.
Project description:The mammalian embryos Caudal Lateral Epiblast (CLE) harbours bipotent progenitors, called Neural Mesodermal Progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here we performed a single cell analysis of different in vitro NMPs populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them to E8.25 CLE mouse embryos. In our analysis of this region our findings challenge the notion that NMPs can be defined by the exclusive coexpression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built Support Vector Machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs, leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs, yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which leads to suggest that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMPs population.