The birth of small-diameter TrkA+ neurons that mediate pain and thermoreception begins approximately 24 hours after the cessation of neural crest cell migration from progenitors residing in the nascent dorsal root ganglion. Although multiple geographically distinct progenitor pools have been proposed, this study is the first to comprehensively characterize the derivation of small-diameter neurons. In the developing chick embryo we identify novel patterns in neural crest cell migration and colonization that sculpt the incipient ganglion into a postmitotic neuronal core encapsulated by a layer of proliferative progenitor cells. Furthermore, we show that this outer progenitor layer is composed of three spatially, temporally, and molecularly distinct progenitor zones, two of which give rise to distinct populations of TrkA+ neurons.

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Developmental, stem cell and cancer biologists are interested in the molecular definition of cellular differentiation. Although single-cell RNA sequencing represents a transformational advance for global gene analyses, novel obstacles have emerged, including the computational management of dropout events, the reconstruction of biological pathways and the isolation of target cell populations. We develop an algorithm named dpath that applies the concept of metagene entropy and allows the ranking of cells based on their differentiation potential. We also develop self-organizing map (SOM) and random walk with restart (RWR) algorithms to separate the progenitors from the differentiated cells and reconstruct the lineage hierarchies in an unbiased manner. We test these algorithms using single cells from Etv2-EYFP transgenic mouse embryos and reveal specific molecular pathways that direct differentiation programmes involving the haemato-endothelial lineages. This software program quantitatively assesses the progenitor and committed states in single-cell RNA-seq data sets in a non-biased manner.

The single cell RNA sequencing (scRNA-seq) technique begin a new era by allowing the observation of gene expression at the single cell level. However, there is also a large amount of technical and biological noise. Because of the low number of RNA transcriptomes and the stochastic nature of the gene expression pattern, there is a high chance of missing nonzero entries as zero, which are called dropout events.

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

Decoding the temporal control of gene expression patterns is key to the understanding of the complex mechanisms that govern developmental decisions during heart development. High-throughput methods have been employed to systematically study the dynamic and coordinated nature of cardiac differentiation at the global level with multiple dimensions. Therefore, there is a pressing need to develop a systems approach to integrate these data from individual studies and infer the dynamic regulatory networks in an unbiased fashion.

All trans retinoic acid (atRA) is one of the most potent therapeutic agents, but extensive toxicity caused by nuclear RA receptors (RARs) limits its clinical application in treating cancer. AtRA also exerts non-genomic activities for which the mechanism remains poorly understood. We determine that cellular retinoic acid binding protein 1 (Crabp1) mediates the non-genomic activity of atRA, and identify two compounds as the ligands of Crabp1 to rapidly and RAR-independently activate extracellular signal regulated kinase 1/2 (ERK1/2). Non-canonically activated ERK activates protein phosphatase 2A (PP2A) and lengthens cell cycle duration in embryonic stem cells (ESC). This is abolished in Crabp1-null ESCs. Re-expressing Crabp1 in Crabp1-negative cancer cells also sensitizes their apoptotic induction by atRA. This study reveals a physiological relevance of the non-genomic action of atRA, mediated by Crabp1, in modulating cell cycle progression and apoptosis induction, and provides a new cancer therapeutic strategy whereby compounds specifically targeting Crabp1 can modulate cell cycle and cancer cell apoptosis in a RAR-independent fashion, thereby avoiding atRA's toxicity caused by its genomic effects.

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.

Remodeling of the primitive vasculature is necessary for the formation of a complex branched vascular architecture. However, the factors that modulate these processes are incompletely defined. Previously, we defined the role of microRNAs (miRNAs) in endothelial specification. In the present study, we further examined the Etv2-Cre mediated ablation of DicerL/L and characterized the perturbed vascular patterning in the embryo proper and yolk-sac. We mechanistically defined an important role for miR-130a, an Etv2 downstream target, in the mediation of vascular patterning and angiogenesis in vitro and in vivo. Inducible overexpression of miR-130a resulted in robust induction of vascular sprouts and angiogenesis with increased uptake of acetylated-LDL. Mechanistically, miR-130a directly regulated Jarid2 expression by binding to its 3'-UTR region. Over-expression of Jarid2 in HUVEC cells led to defective tube formation indicating its inhibitory role in angiogenesis. The knockout of miR-130a showed increased levels of Jarid2 in the ES/EB system. In addition, the levels of Jarid2 transcripts were increased in the Etv2-null embryos at E8.5. In the in vivo settings, injection of miR-130a specific morpholinos in zebrafish embryos resulted in perturbed vascular patterning with reduced levels of endothelial transcripts in the miR-130a morphants. Further, co-injection of miR-130a mimics in the miR-130a morphants rescued the vascular defects during embryogenesis. qPCR and in situ hybridization techniques demonstrated increased expression of jarid2a in the miR-130a morphants in vivo. These findings demonstrate a critical role for Etv2-miR-130a-Jarid2 in vascular patterning both in vitro and in vivo.

The mammalian heart has a limited regenerative capacity and typically progresses to heart failure following injury. Here, we defined a hedgehog (HH)-Gli1-Mycn network for cardiomyocyte proliferation and heart regeneration from amphibians to mammals. Using a genome-wide screen, we verified that HH signaling was essential for heart regeneration in the injured newt. Next, pharmacological and genetic loss- and gain-of-function of HH signaling demonstrated the essential requirement for HH signaling in the neonatal, adolescent, and adult mouse heart regeneration, and in the proliferation of hiPSC-derived cardiomyocytes. Fate-mapping and molecular biological studies revealed that HH signaling, via a HH-Gli1-Mycn network, contributed to heart regeneration by inducing proliferation of pre-existing cardiomyocytes and not by de novo cardiomyogenesis. Further, Mycn mRNA transfection experiments recapitulated the effects of HH signaling and promoted adult cardiomyocyte proliferation. These studies defined an evolutionarily conserved function of HH signaling that may serve as a platform for human regenerative therapies.

Mechanisms of haematopoietic and cardiac patterning remain poorly understood. Here we show that the BMP and Wnt signalling pathways are integrated in an endoglin (Eng)-dependent manner in cardiac and haematopoietic lineage specification. Eng is expressed in early mesoderm and marks both haematopoietic and cardiac progenitors. In the absence of Eng, yolk sacs inappropriately express the cardiac marker, Nkx2.5. Conversely, high levels of Eng in vitro and in vivo increase haematopoiesis and inhibit cardiogenesis. Levels of Eng determine the activation of both BMP and Wnt pathways, which are integrated downstream of Eng by phosphorylation of Smad1 by Gsk3. By interrogating Eng-dependent Wnt-mediated transcriptional changes, we identify Jdp2 as a key Eng-dependent Wnt target, sufficient to establish haematopoietic fate in early mesoderm when BMP and Wnt crosstalk is disturbed. These studies provide mechanistic insight into the integration of BMP and Wnt signalling in the establishment of haematopoietic and cardiac progenitors during embryogenesis.

Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.

Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP(+) cells are derived from Mesp1-Cre(+) cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.

Profiling single cell gene expression data over specified time periods are increasingly applied to the study of complex developmental processes. Here, we describe a novel prototype-based dimension reduction method to visualize high throughput temporal expression data for single cell analyses. Our software preserves the global developmental trajectories over a specified time course, and it also identifies subpopulations of cells within each time point demonstrating superior visualization performance over six commonly used methods.