Guangdun Peng

Spatial Transcriptome for the Molecular Annotation of Lineage Fates and Cell Identity in Mid-gastrula Mouse Embryo

Gastrulation of the mouse embryo entails progressive restriction of lineage potency and the organization of the lineage progenitors into a body plan. Here we performed a high-resolution RNA sequencing analysis on single mid-gastrulation mouse embryos to collate a spatial transcriptome that correlated with the regionalization of cell fates in the embryo. 3D rendition of the quantitative data enabled the visualization of the spatial pattern of all expressing genes in the epiblast in a digital whole-mount in situ format. The dataset also identified genes that (1) are co-expressed in a specific cell population, (2) display similar global pattern of expression, (3) have lineage markers, (4) mark domains of transcriptional and signaling activity associated with cell fates, and (5) can be used as zip codes for mapping the position of single cells isolated from the mid-gastrula stage embryo and the embryo-derived stem cells to the equivalent epiblast cells for delineating their prospective cell fates.

The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways

The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positive transcription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. In this study, we showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. Our results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals. DOI: http://dx.doi.org/10.7554/eLife.02224.001

SIP30 is regulated by ERK in peripheral nerve injury-induced neuropathic pain

ERK plays an important role in chronic neuropathic pain. However, the underlying mechanism is largely unknown. Here we show that in chronic constriction injury-treated rat spinal cords, up-regulation of SIP30 (SNAP25-interacting protein 30), which is involved in the development and maintenance of chronic constriction injury-induced neuropathic pain, correlates with ERK activation and that the up-regulation of SIP30 is suppressed by intrathecal delivery of the MEK inhibitor U0126. In PC12 cells, up-regulation of SIP30 by nerve growth factor is also dependent on ERK activation. We found that there is an ERK-responsive region in the rat sip30 promoter. Activation of ERK promotes the recruitment of the transcription factor cyclic AMP-response element-binding protein to the sip30 gene promoter. Taken together, our results provide a potential downstream target of ERK activation-mediated neuropathic pain.

Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq

Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously. The Geo-seq protocol allows the profiling of transcriptome information from only a small number cells and retains their native spatial information. This protocol has wide potential applications to address biological and pathological questions of cellular properties such as prospective cell fates, biological function and the gene regulatory network. Geo-seq has been applied to investigate the spatial transcriptome of mouse early embryo, mouse brain, and pathological liver and sperm tissues. The entire protocol from tissue collection and microdissection to sequencing requires ∼5 d, Data analysis takes another 1 or 2 weeks, depending on the amount of data and the speed of the processor.

Histone deacetylation promotes mouse neural induction by restricting Nodal-dependent mesendoderm fate.

Cell fate determination requires the cooperation between extrinsic signals and intrinsic molecules including transcription factors as well as epigenetic regulators. Nevertheless, how neural fate commitment is regulated by epigenetic modifications remains largely unclear. Here we show that transient histone deacetylation at epiblast stage promotes neural differentiation of mouse embryonic stem cells (mESCs). Histone deacetylase 1 (HDAC1) deficiency in mESCs partially phenocopies the inhibition of histone deacetylation in vitro, and displays reduced incorporation into neural tissues in chimeric mouse embryos in vivo. Mechanistic studies show that Nodal, which is repressed by histone deacetylation, is a direct target of HDAC1. Furthermore, the inhibition of histone deacetylation in the anterior explant of mouse embryos at E7.0 leads to Nodal activation and neural development repression. Thus, our study reveals an intrinsic mechanism that epigenetic histone deacetylation ensures neural fate commitment by restricting Nodal signalling in murine anterior epiblast ex vivo and mESC in vitro.

Role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain

ABSTRACT Using the chronic constriction injury (CCI) model of neuropathic pain, we profiled gene expression in the rat spinal cord, and identified SIP30 as a gene whose expression was elevated after CCI. SIP30 was previously shown to interact with SNAP25, but whose function was otherwise unknown. We now show that in the spinal cord, SIP30 was present in the dorsal horn laminae where the peripheral nociceptive inputs first synapse, co‐localizing with nociception‐related neuropeptides CGRP and substance P. With the onset of neuropathic pain after CCI surgery, SIP30 mRNA and protein levels increased in the ipsilateral side of the spinal cord, suggesting a potential association between SIP30 and neuropathic pain. When CCI‐upregulated SIP30 was inhibited by intrathecal antisense oligonucleotide administration, neuropathic pain was attenuated. This neuropathic pain‐reducing effect was observed both during neuropathic pain onset following CCI, and after neuropathic pain was fully established, implicating SIP30 involvement in the development and maintenance phases of neuropathic pain. Using a secretion assay in PC12 cells, anti‐SIP30 siRNA decreased the total pool of synaptic vesicles available for exocytosis, pointing to a potential function for SIP30. These results suggest a role of SIP30 in the development and maintenance of peripheral nerve injury‐induced neuropathic pain.

Intrinsic regulations in neural fate commitment.

Neural fate commitment is an early embryonic event that a group of cells in ectoderm, which do not ingress through primitive streak, acquire a neural fate but not epidermal or mesodermal lineages. Several extracellular signaling pathways initiated by the secreted proteins bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), wingless/int class proteins (WNTs) and Nodal play essential roles in the specification of the neural plate. Accumulating evidence from the studies on mouse and pluripotent embryonic stem cells reveals that except for the extracellular signals, the intracellular molecules, including both transcriptional and epigenetic factors, participate in the modulation of neural fate commitment as well. In the review, we mainly focus on recent findings that the initiation of the nervous system is elaborately regulated by the intrinsic programs, which are mediated by transcriptional factors such as Sox2, Zfp521, Sip1 and Pou3f1, as well as epigenetic modifications, including histone methylation/demethylation, histone acetylation/deacetylation, and DNA methylation/demethylation. The discovery of the intrinsic regulatory machineries provides better understanding of the mechanisms by which the neural fate commitment is ensured by the cooperation between extracellular factors and intracellular molecules.

Ectodermal progenitors derived from epiblast stem cells by inhibition of Nodal signaling

The ectoderm has the capability to generate epidermis and neuroectoderm and plays imperative roles during the early embryonic development. Our recent study uncovered a region with ectodermal progenitor potential in mouse embryo at embryonic day 7.0 and revealed that Nodal inhibition is essential for its formation. Here, we demonstrate that through brief inhibition of Nodal signaling in vitro, mouse embryonic stem cell (ESC)-derived epiblast stem cells (ESD-EpiSCs) could be committed to transient ectodermal progenitor populations, which possess the ability to give rise to neural or epidermal ectoderm in the absence or presence of BMP4, respectively. Mechanistic studies reveal that BMP4 recruits distinct transcriptional targets in ESD-EpiSCs and ectoderm-like cells. Furthermore, FGF-Erk signaling may also be alleviated during the generation of ectoderm-like cells. Thus, our data suggest that instructive interactions among several extracellular signals participate in the commitment of ectoderm from ESD-EpiSCs, which shed new light on the understanding of the formation of ectoderm during the gastrulation in early mouse embryo development.

Dynamic Heterogeneity of Brachyury in Mouse Epiblast Stem Cells Mediates Distinct Response to Extrinsic Bone Morphogenetic Protein (BMP) Signaling

Mouse pluripotent cells, such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), provide excellent in vitro systems to study imperative pre- and postimplantation events of in vivo mammalian development. It is known that mouse ESCs are dynamic heterogeneous populations. However, it remains largely unclear whether and how EpiSCs possess heterogeneity and plasticity similar to that of ESCs. Here, we show that EpiSCs are discriminated by the expression of a specific marker T (Brachyury) into two populations. The T-positive (T+) and the T-negative (T−) populations can be interconverted within the same culture condition. In addition, the two populations display distinct responses to bone morphogenetic protein (BMP) signaling and different developmental potentials. The T− EpiSCs are preferentially differentiated into ectoderm lineages, whereas T+ EpiSCs have a biased potential for mesendoderm fates. Mechanistic studies reveal that T+ EpiSCs have an earlier and faster response to BMP4 stimulation than T− EpiSCs. Id1 mediates the commitment of T− EpiSCs to epidermal lineage during BMP4 treatment. On the other hand, Snail modulates the conversion of T+ EpiSCs to mesendoderm fates with the presence of BMP4. Furthermore, T expression is essential for epithelial-mesenchymal transition during EpiSCs differentiation. Our findings suggest that the dynamic heterogeneity of the T+/T− subpopulation primes EpiSCs toward particular cell lineages, providing important insights into the dynamic development of the early mouse embryo.

Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta

Novel regenerative therapies may stem from deeper understanding of the mechanisms governing cardiovascular lineage diversification. Using enhancer mapping and live imaging in avian embryos, and genetic lineage tracing in mice, we investigated the spatio-temporal dynamics of cardiovascular progenitor populations. We show that expression of the cardiac transcription factor Nkx2.5 marks a mesodermal population outside of the cardiac crescent in the extraembryonic and lateral plate mesoderm, with characteristics of hemogenic angioblasts. Extra-cardiac Nkx2.5 lineage progenitors migrate into the embryo and contribute to clusters of CD41+/CD45+ and RUNX1+ cells in the endocardium, the aorta-gonad-mesonephros region of the dorsal aorta and liver. We also demonstrated that ectopic expression of Nkx2.5 in chick embryos activates the hemoangiogenic gene expression program. Taken together, we identified a hemogenic angioblast cell lineage characterized by transient Nkx2.5 expression that contributes to hemogenic endothelium and endocardium, suggesting a novel role for Nkx2.5 in hemoangiogenic lineage specification and diversification. DOI: http://dx.doi.org/10.7554/eLife.20994.001