Bipotent Progenitors Research Articles

Epigenetic and Single Cell Analyses Reveal Dual Fate Decision Points in Pre-Primed Hematopoietic Stem Cells during Erythro-Megakaryopoiesis

Recent single cell analyses have shown that hematopoietic differentiation is more complex than the traditional hierarchical model suggests, and that lineage commitment may occur earlier in the hierarchy than previously thought (Notta, 2016; Psaila, 2017). In particular, the relationship between bipotential megakaryocyte-erythroid progenitors (MEP) and common myeloid progenitors (CMP) is unclear in erythro-megakaryopoiesis. While it has been established that mouse MEP and CMP populations contain different proportions of megakaryocytic and erythroid lineage-committed cells, our aim was to determine when lineage-commitment occurs and whether a ‘default’ differentiation pathway exists.We established the transcriptional and epigenetic profiles of 14 flow cytometry-enriched primary murine hematopoietic populations, including hematopoietic stem/progenitor cells (HSPC) and lineage-committed populations. We performed transcriptome profiling (RNASeq), chromatin accessibility profiling (Assay for Transposable-Accessible Chromatin; ATACSeq), and DNA methylation profiling (MethylCSeq). We also performed ChIPSeq of H3K27ac on erythroblasts (EB) and immature megakaryocytes (Mk) to identify putative active enhancer elements.Principle component analysis (PCA) of the RNASeq data demonstrated variation between lineages. Primary cells and cell lines were separated by PC1 (20% of variance) while PC2 (14% of variance) separated HSPC and Mk-committed cells from EB, monocytes and granulocytes. Likewise, PCA of ATACSeq data also indicated that HSPC and Mk shared more similarity than other populations: PC1 (19% of variance) separated EB and non-EB populations while PC2 (12% of variance) separated HSPC and Mk-committed cells from EB, monocytes, granulocytes and lymphocytes (Figure 1). These data demonstrate that the transcriptional and chromatin accessibility profiles of Mk are more similar to HSPC than to other populations.We defined putative active enhancers (pAE) as the intersection of ATACSeq and H3K27ac ChIPSeq peaks in EB and Mk. We identified 2098 EB- and 6386 Mk-specific pAE, and 5989 shared pAE. To predict pAE in progenitors we overlaid EB and Mk pAE with successive ATACSeq peaks in lineage-restricted progenitors (CFU-E, CFU-Mk), followed by CMP and the Lin-Sca+Kit+ population (LSK). 97% and 89% of Mk pAE were detected in CMP and LSK, whereas only 84% and 64% of EB pAE were detected. To validate which EB and Mk pAE in LSK were active (as opposed to poised), we compared our LSK pAE with the indexing-first ChIP histone marks identified by Lara-Astiaso et al., (Science , 2014). We found that 31% of Mk-, but only 13% of EB pAE, were active in the LSK. These data indicate that the MK enhancer profile is established earlier than that of EB.Our comparison of the DNA methylation (CH3) profiles showed that, unlike the RNASeq, ATACSeq, and H3K27ac profiles, erythropoiesis was associated with an 87% decrease in CH3 from CMP and LSK sets; 15% of EB CH3 peaks were established de novo. In contrast, megakaryopoiesis was associated with a 33% increase in CH3 peaks from CMP and LSK sets; 50% of Mk CH3 peaks were established de novo. Many of these de novo methylation sites were associated with genes directing non-Mk lineages that are downregulated compared to CMP (e.g., Dach2, Sox5, Kit, Klf1, Tet1, Fog1).To determine when lineage-commitment occurs we performed single cell RNASeq (10X Genomics platform) on ~9000 cells from each of LSK, CMP, MEP, and GMP (granulocyte-macrophage progenitors) populations. The Seurat software (Sajita, 2015) was used to ‘cluster’ cells with similar expression profiles. MEP contained 5 erythroid clusters of different maturation stages (57% of cells), and 1 cluster with an Mk gene signature (5% of cells). CMP contained, among others, 8 Mk clusters (60% of cells) and 1 cluster (2% of cells) co-expressing Mk and EB genes. LSK contained 4 Mk clusters (26% of cells) but no populations expressing EB-associated genes.We conclude that the transcriptional and epigenetic Mk profile is detectable at the earliest stages of hematopoiesis. The requirement of de novo methylation and silencing of other lineage-associated factors prevents the classification of Mk as a default pathway. In contrast, the EB profile is established after most megakaryocyte progenitor cells have become committed and extensive transcriptional and epigenetic reprogramming is required to permit erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Prostaglandin E2 Regulates Bipotent Monocyte-Dendritic Progenitor Cell Lineage-Commitment.

The factors/mechanisms regulating multipotent or bipotent hematopoietic progenitor cells lineage-commitment are not well understood. In this study, we found that prostaglandin E2 (PGE2) is a crucial physiological regulator of lineage choice for the bipotential monocyte-dendritic progenitor cell (MDP). Inhibition of endogenous PGE2 biosynthesis in mice by the dual cyclooxygenase inhibitor, indomethacin, enhances bone marrow and spleen monocyte (MO) differentiation and reduces dendritic cell (DC) differentiation. Ex vivo treatment of purified MDP with indomethacin preferentially increases MO development at the expense of DC generation, whereas addition of exogenous PGE2 reverses the indomethacin-mediated alteration in MDP differentiation potential. Treatment of MDP with selective EP receptor agonists demonstrated that EP1 signaling promotes MDP differentiation into DC at the expense of MO generation. Conversely, EP1 receptor knockout mice showed reduced DC and increased MO differentiation. Mechanistic studies revealed that PGE2 increases expression of the tyrosine kinase receptor Flt3 on MDP and increases the DC-lineage-related transcription factor PU.1, while reducing expression of M-CSFR and the MO-lineage-related transcription factor MafB. These data indicate that PGE2-EP1 signaling plays a critical role in MDP lineage commitment and DC and MO differentiation.

The conserved sex regulator DMRT1 recruits SOX9 in sexual cell fate reprogramming.

Mammalian sexual development commences when fetal bipotential progenitor cells adopt male Sertoli (in XY) or female granulosa (in XX) gonadal cell fates. Differentiation of these cells involves extensive divergence in chromatin state and gene expression, reflecting distinct roles in sexual differentiation and gametogenesis. Surprisingly, differentiated gonadal cell fates require active maintenance through postnatal life to prevent sexual transdifferentiation and female cell fate can be reprogrammed by ectopic expression of the sex regulator DMRT1. Here we examine how DMRT1 reprograms granulosa cells to Sertoli-like cells in vivo and in culture. We define postnatal sex-biased gene expression programs and identify three-dimensional chromatin contacts and differentially accessible chromatin regions (DARs) associated with differentially expressed genes. Using a conditional transgene we find DMRT1 only partially reprograms the ovarian transcriptome in the absence of SOX9 and its paralog SOX8, indicating that these factors functionally cooperate with DMRT1. ATAC-seq and ChIP-seq show that DMRT1 induces formation of many DARs that it binds with SOX9, and DMRT1 is required for binding of SOX9 at most of these. We suggest that DMRT1 can act as a pioneer factor to open chromatin and allow binding of SOX9, which then cooperates with DMRT1 to reprogram sexual cell fate.

Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum.

Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.

Context-aware synthetic biology by controller design: Engineering the mammalian cell.

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications.

Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation

SummaryThe extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

Emergence and Patterning Dynamics of Mouse Definitive Endoderm

The segregation of definitive endoderm (DE) from bi-potent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a novel dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in a few isolated cells in vivo within a temporally restricted window. Using 2D and 3D in vitro models, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

Spatiotemporal contribution of neuromesodermal progenitor-derived neural cells in the elongation of developing mouse spinal cord

AimsDuring vertebrate development, the posterior end of the embryo progressively elongates in a head-to-tail direction to form the body plan. Recent lineage tracing experiments revealed that bi-potent progenitors, called neuromesodermal progenitors (NMPs), produce caudal neural and mesodermal tissues during axial elongation. However, their precise location and contribution to spinal cord development remain elusive. Main methodsHere we used NMP-specific markers (Sox2 and BraT) and a genetic lineage tracing system to localize NMP progeny in vivo. Key findingsSox2 and BraT double positive cells were initially located at the tail tip, but were later found in the caudal neural tube, which is a unique feature of mouse development. In the neural tube, they produced neural progenitors (NPCs) and contributed to the spinal cord gradually along the AP axis during axial elongation. Interestingly, NMP-derived NPCs preferentially contributed to the ventral side first and later to the dorsal side at the lumbar spinal cord level, which may be associated with atypical junctional neurulation in mice. SignificanceOur current observations detail the contribution of NMP progeny to spinal cord elongation and provide insights into how different species uniquely execute caudal morphogenesis.

Mesenchymal Stem Cells (MSCs) in Targeted Drug Delivery: Literature Review and Exploratory Data on Migrating and Differentiation Capacities of Bone MSCs into Hepatic Progenitor Cells.

Mesenchymal stem cells (MSCs), particularly bone MSCs (BMSCs) offer great potentials for targeted therapeutic applications owing to their migratory and differentiation capacities. Significant advances have been achieved in the differentiation of hepatocyte or hepatocyte-like cells both in vitro and in vivo. However, there is limited knowledge on the differentiation of BMSCs into bipotential hepatic progenitor cells or cholangiocyte. This study reviews the potentials and advances in using MSCs as vehicles for targeted drug delivery and proposes a new method for the induction of differentiation in rat BMSCs into hepatic progenitor cells in vitro and assesses the differential and migratory capacities. The BMSCs of Sprague Dawley (SD) rats were harvested from the femur and the tibiae of the rats. After isolation and culturing, BMSCs from Passage 1 were used for the study. The in vitro differentiation of the hepatic progenitor cells was performed using a 2-step induction approach after 5-day serum deprivation from the BMSCs and culturing in Dulbecco's modified eagle medium. Spontaneous in vitro differentiation of BMSCs was examined in the absence of growth factors for 15 days as control treatment. Hepatocytes differentiation was achieved by exposing the culture to collagen type I-coated plates. Cholangiocytes differentiation was achieved with replating the BMC-HepPCs on a layer of Matrigel. Immunofluorescence was conducted on twelve-well plates to determine cell differentiations. Real-Time Quantitative Reverse Transcription PCR (qRTPCR) was used to determine the total RNA extracted using the Trizol LS reagent. In the hepatocyte differentiation group, after periodic acid-schiff (PAS) staining for glycogen, inverted microscope was used to determine differentiations and undifferentiated BMC-HepPCs served as controls. The amount of low-density lipoprotein (LDL) uptake by the BMSCs-derived hepatocytes was assessed using fluorescence microscopy. The secretion of rat albumin was quantified using a quantitative ELISA kit. Differentiation induction is indicative of the sequential supplementation of sodium butyrate and cytokines, which are involved in the embryonic development of the mammalian liver. Hepatic progenitor cells, derived from bone marrow, can be differentiated bidirectionally in vitro into both hepatocyte and cholangiocyte cell-lines. The differentiated cells, including hepatic progenitor cells, hepatocytes, and bile duct-like cells, were identified and analyzed at mRNA and protein levels. Our findings show that BMSCs can be utilized as novel bipotential hepatic progenitor cells and thereby for hepatobiliary disease treatment or hepatobiliary tissue-engineering.

MET and EGFR signaling pathways play different roles in bile duct ligation (BDL) mice by modulating distinct cell responses

In previous studies we have demonstrated the critical roles of EGFR and HGFR (c-Met) for liver regeneration and for conversion of hepatocytes to cholangiocytes by a “clean” partial hepatectomy model, in which hepatocytes and cholangiocytes grow rapidly and synchronously. In contrast, vast majority of liver diseases involve a prolonged pathologic duration, therefore multiple types of cells participate and play different roles in the variant stages. However, it is not fully understood that how EGFR and MET play their roles in this complicated setting. In this study, we explore the role of MET and EGFR signaling pathways in biliary injury after common bile duct ligation. Mice with inhibition of EGFR signaling alone (EGFRi) showed abolished hepatocyte proliferation and biliary duct cell proliferation 2, 5 and 7 days from Ki67 immunochemistry staining. However, MET knockout mice (KO) showed increased proliferation of cholangiocytes at 2, 5 and 7 days. MET KO mice subjected to BDL surgery had an increase of the CK19 marked ductular reaction at 2, 5 and 7 days. In contrast, EGFRi mice showed a reduced ductular reaction at the same time point. There was enhanced immunohistochemistry for Sox9, in EGFRi mice, but absence of its expression in MET KO mice at 7 days post BDL. From western blot results of total liver tissue lysate, we found increased EpCAM expression after BDL at 7 days in MET KO but not in EGFRi mice. Interestingly, we also observed that both p-Merlin and cleaved p-Mst1, components of the Hippo pathway, decreased only in Met knockout mice. Mice with combined disruption of MET and EGFR signaling became very ill by 24 hours and died at 1 to 2 days after bile duct ligation. From Kaplan-Meier analysis, both EGFR inhibition and Met knockout mice had decreased survival rate starting at 11 days, though the survival rate of MET KO mice was overall much better than EGFRi mice by around 40%. We also observed that p-Ezrin (Thr567) was downregulated in either MET KO or EGFRi mice, while the total Ezrin expression did not change. Conclusions Our study demonstrates that HGFR (MET) and EGFR control biliary response to bile duct ligation. The cholangiocyte responses, however, were very different between these two receptors. Their combined disruption led to acute liver failure and early death following bile duct ligation. The receptor tyrosine kinases c-Met and EGFR may play different roles in the biliary injury-repair responses, including proliferation, trans-differentiation and bi-potential progenitor cells activation.

Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages.

Toll-like receptor 5 (TLR5) is the receptor of bacterial Flagellin. Reportedly, TLR5 engagement helps to combat infections, especially at mucosal sites, by evoking responses from epithelial cells and immune cells. Here we report that TLR5 is expressed on a previously defined bipotent progenitor of macrophages (MΦs) and osteoclasts (OCs) that resides in the mouse bone marrow (BM) and circulates at low frequency in the blood. In vitro, Flagellin promoted the generation of MΦs, but not OCs from this progenitor. In vivo, MΦ/OC progenitors were recruited from the blood into the lung upon intranasal inoculation of Flagellin, where they rapidly differentiated into MΦs. Recruitment of the MΦ/OC progenitors into the lung was likely promoted by the CCL2/CCR2 axis, since the progenitors expressed CCR2 and type 2 alveolar epithelial cells (AECs) produced CCL2 upon stimulation by Flagellin. Moreover, CCR2 blockade reduced migration of the MΦ/OC progenitors toward lung lavage fluid (LLF) from Flagellin-inoculated mice. Our study points to a novel role of the Flagellin/TLR5 axis in recruiting circulating MΦ/OC progenitors into infected tissue and stimulating these progenitors to locally differentiate into MΦs. The progenitor pathway to produce MΦs may act, next to monocyte recruitment, to fortify host protection against bacterial infection at mucosal sites.

Chemical conversion of aged hepatocytes into bipotent liver progenitor cells.

In the aging society, understanding the influence of hepatocyte age on hepatocyte donation may inform efforts to expand alternative cell sources to mitigate liver donor shortage. A combination of the molecules Y27632, A-83-01, and CHIR99021 has been used to reprogram rodent young hepatocytes into chemically induced liver progenitor (CLiP) cells; however, whether it could also reprogram aged hepatocytes has not yet been elucidated. Primary hepatocytes were isolated from aged and young donor rats, respectively. Hepatic histological changes were evaluated. Differences in gene expression in hepatocytes were identified. The in vitro reprogramming plasticity of hepatocytes as evidenced by CLiP conversion and the hepatocyte and cholangiocyte maturation capacity of reprogrammed CLIPs were analyzed. The effect of hepatocyte growth factor (HGF) on cell propagation was also investigated. The histological findings revealed ongoing liver damage with inflammation, fibrosis, senescence, and ductular reaction in aged livers. Microarray analysis showed altered gene expression profiles in hepatocytes from aged donors, especially with regard to metabolic pathways. Aged hepatocytes could be converted into CLiPs (Aged-CLiPs) expressing progenitor cell markers, but with a relatively low proliferative rate compared with young hepatocytes. Aged-CLiPs possessed both hepatocyte and cholangiocyte maturation capacity. HGF facilitated CLiP conversion in aged hepatocytes, which was partly related to the activation of Erk1 and Akt1 signaling. Aged rat hepatocytes have retained reprogramming plasticity as evidenced by CLiP conversion in culture. HGF promoted proliferation and CLiP conversion in aged hepatocytes. Hepatocytes from aged donors may be used as an alternative cell source to mitigate donor shortage.

Neuromesodermal Progenitors: A Basis for Robust Axial Patterning in Development and Evolution.

During early development the vertebrate embryo elongates through a combination of tissue shape change, growth and progenitor cell expansion across multiple regions of the body axis. How these events are coordinated across the length of the embryo to generate a well-proportioned body axis is unknown. Understanding the multi-tissue interplay of morphogenesis, growth and cell fate specification is essential for us to gain a complete understanding how diverse body plans have evolved in a robust manner. Within the posterior region of the embryo, a population of bipotent neuromesodermal progenitors generate both spinal cord and paraxial mesoderm derivatives during the elongation of the vertebrate body. Here we summarize recent data comparing neuromesodermal lineage and their underlying gene-regulatory networks between species and through development. We find that the common characteristic underlying this population is a competence to generate posterior neural and paraxial mesoderm cells, with a conserved Wnt/FGF and Sox2/T/Tbx6 regulatory network. We propose the hypothesis that by maintaining a population of multi-germ layer competent progenitors at the posterior aspect of the embryo, a flexible pool of progenitors is maintained whose contribution to the elongating body axis varies as a consequence of the relative growth rates occurring within anterior and posterior regions of the body axis. We discuss how this capacity for variation in the proportions and rates of NM specification might have been important allowing for alterations in the timing of embryo growth during evolution.

Lnc-RHL, a novel long non-coding RNA required for the differentiation of hepatocytes from human bipotent progenitor cells.

ObjectivesThe final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc‐RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes.Materials and MethodsDifferentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT‐PCR and RACE, we cloned a novel lncRNA (lnc‐RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc‐RHL concurrently with differentiation, we show that lnc‐RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage.ResultsHere, we report the discovery of lnc‐RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc‐RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc‐RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes.ConclusionsDeficiency for lnc‐RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc‐RHL is a key regulator of bipotency in HepaRG cells.

Neuromesodermal Progenitor Origin of Trunk Neural Crest <i>in vivo</i>

Neural crest (NC) is a vertebrate-specific population of multipotent embryonic cells predisposed to diverse derivatives along the anteroposterior (A-P) axis. Only cranial NC progenitors give rise to ectomesenchymal cell types, whereas trunk NC is biased for neuronal cell fates. By integrating multimodal single-cell analysis, we provide evidence for divergent embryonic origins of cranial vs. trunk NC that explain this dichotomy. We show that the NC regulator neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning is heterogeneously expressed across the A-P axis and identify its specific cranial and trunk autoregulatory enhancers. Whereas cranial-specific enhancer is active in the bona fide NC, the trunk neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning autoregulatory element surprisingly marked bipotent tailbud neuromesodermal progenitors (NMps). We integrated NMp single cell epigemomics and trasncriptomics data and for the first time reconstructed anamniote NMp gene regulatory network. Moreover, using pseudotime and developmental trajectory analyses of NMps and NC during normal development and inneural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning mutants, we demonstrate an active role for neural crest, neuromesodermal progenitors, gene regulatory network, axial progenitors, embryo patterning in balancing non-cranial NC and NMp fates during early embryonic development. Strikingly, we show that a portion of posterior NC in the developing zebrafish embryo is derived from the pro-neural NMps. This suggests a common embryonic origin of trunk NC and NM progenitors that is distinct from cranial NC anlage, and elucidates pro-neural bias of trunk NC.

Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation.

Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes-expressing progenitors in response to different Nodal signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single-cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes-dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes-positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggested the immediate and complete separation of AM and DE lineages from Eomes-expressing cells as last common bipotential progenitor.

Two Sequential and Independent Pathways of Erythromyeloid Progenitor Commitment in Their Niche of Emergence

The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocyte/granulocytes, mast cells and macrophages, the latter in a Myb-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from bipotent progenitors and is absent in Myb-deficient embryos. We identified Csf2rb as a novel marker of these Myb-dependent megakaryocyte-erythroid bipotent progenitors that colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes.

The people behind the papers - Zhi Ye and David Kimelman.

The anterior to posterior extension of the vertebrate body axis relies on a population of bipotent neuromesodermal progenitors in the tailbud. A new paper in Development uncovers a crucial and unexpected new role for Hox13 genes in sustaining these progenitors to promote axis extension in zebrafish. To hear more about the story, we caught up with the paper's two authors: postdoctoral researcher Zhi Ye and his supervisor David Kimelman, Professor of Biochemistry and Adjunct Professor of Biology at the University of Washington, Seattle.