The mammalian thalamus is located in the diencephalon and is composed of dozens of morphologically and functionally distinct nuclei. The majority of these nuclei project axons to the neocortex in unique patterns and play critical roles in sensory, motor, and cognitive functions. It has been assumed that the adult thalamus is derived from neural progenitor cells located within the alar plate of the caudal diencephalon. Nevertheless, how a distinct array of postmitotic thalamic nuclei emerge from this single developmental unit has remained largely unknown. Our recent studies found that these thalamic nuclei are in fact derived from molecularly heterogeneous populations of progenitor cells distributed within at least two distinct progenitor domains in the caudal diencephalon. In this study, we investigated how such molecular heterogeneity is established and maintained during early development of the thalamus and how early signaling mechanisms influence the formation of postmitotic thalamic nuclei. By using mouse genetics and in utero electroporation, we provide evidence that Sonic hedgehog (Shh), which is normally expressed in ventral and rostral borders of the embryonic thalamus, plays a crucial role in patterning progenitor domains throughout the thalamus. We also show that increasing or decreasing Shh activity causes dramatic reorganization of postmitotic thalamic nuclei through altering the positional identity of progenitor cells.
Vasculogenesis/angiogenesis is one of the earliest processes that occurs during embryogenesis. ETV2 and SOX7 were previously shown to play a role in endothelial development; however, their mechanistic interaction has not been defined. In the present study, concomitant expression of Etv2 and Sox7 in endothelial progenitor cells was verified. ETV2 was shown to be a direct upstream regulator of Sox7 that binds to ETV2 binding elements in the Sox7 upstream regulatory region and activates transcription. We observed that SOX7 over-expression can mimic ETV2 and increase endothelial progenitor cells in embryonic bodies (EBs), while knockdown of Sox7 is able to block ETV2-induced increase in endothelial progenitor cell formation. Angiogenic sprouting was increased by ETV2 over-expression in EBs, and it was significantly decreased in the presence of Sox7 shRNA. Collectively, these studies support the conclusion that ETV2 directly regulates Sox7, and that ETV2 governs endothelial development by regulating transcriptional networks which include Sox7.
Etv2 has recently been shown to be an important factor for the specification of the endocardial/endothelial and hematopoietic lineages. Transgenic embryos lacking Etv2 die between E9.0 and E9.5 due to absence of the endocardial/endothelial lineage and significant disruption of hematopoietic lineages. Sox 7, Sox 17 and Sox 18 comprise the Sox-F family which has been shown to be important in the regulation of hematopoietic cells and cardiovascular development. Therefore, we examined the hypothesis that the Sox family members were downstream targets of Etv2. Sox 7, Sox 17 and Sox 18 were found to be significantly downregulated in the Etv2 null mice compared to wildtype (WT) littermates at two developmental stages. WT embryoid body (EB) developmental expression profiles supported that Sox 7 and Sox 17, but not Sox 18, were direct downstream targets of Etv2. Studies performed using ES cells generated from the Etv2 null and WT littermate blastocysts confirmed the lack of the endothelial and hemogenic endothelium lineages in the absence of Etv2. The expression of all Sox F family members was also markedly altered in the Etv2 null EB system with a virtual absence of both Sox 7 and Sox18 expression in all developmental timepoints in the Etv2 null EBs when compared to WT controls. We utilized an array of molecular biological techniques to demonstrate that Etv2 is a direct upstream regulator of the Sox 7 gene. We also generated an inducible Sox7 overexpressing embryonic stem (ES) cell line and overexpression of Sox7 resulted in an induction of the endothelial molecular program with an increase in the percentage of Flk-1/Cdh5 double positive endothelial progenitor cells and increased expression of Pecam1 and Cdh5 in Sox7 overexpressing Day6 EBs. Collectively, these studies support the conclusion that Etv2 is essential for the endocardial/endothelial lineage including the hemogenic endothelium and Sox7 is an important factor in this developmental pathway. To further verify the role of Sox 7, we are undertaking rescue experiments of the Etv2 null ES cells by expressing Sox 7 under the control of an inducible lentivirus system. We anticipate that the induction of Sox 7 in Etv2 null EBs will reestablish expression of the endothelial and hemogenic endothelial molecular programs.
Background: The molecular definition of the differentiation process is of intense interest for cardiovascular developmental biologists. With the advent of single cell technologies, a number of a transcriptomic approaches have been developed to obtain a complete description of gene expression. While single cell RNA sequencing is a significant advance for describing gene expression at the cellular resolution, novel obstacles have emerged such as computational management of dropout events, discovery of the gene regulatory modules responsible for lineage specification and identification of novel lineage markers. Methods: Using Etv2- and Nkx2-5 EYFP transgenic embryos, from which progenitors of hematopoietic, endothelial and cardiac lineages can be isolated with high purity, we performed single cell transcriptome analysis. We developed a Bayesian statistical framework to (1) model the dropout events, (2) reconstruct the lineage specification pathways from mesodermal progenitors to hematopoietic, endothelial and cardiac lineages, (3) discover the core gene regulatory modules that govern lineage specification and (4) predict novel cell surface markers for cardiac components. We developed a structural stochastic variational inference method to efficiently estimate the model parameters for sequencing data with thousands of cells. Results: The analysis of 421 Etv2- and Nkx2-5 EYFP positive cells predicted the core regulatory networks involving transcription factors, signaling molecules and cell surface proteins, where the signaling cascades are significantly correlated the specification of the endothelial, hematopoietic and various cardiac lineages from the mesodermal progenitors. The method also predicted novel cell surface markers for the first and second heart fields. Conclusions: We report the analysis of complete gene expression profiles of mesodermal lineage specification at single cell resolution. We provide the first unified statistical framework to systematically analyze single cell RNA-seq data and quantitatively couple the network analysis and lineage specification. This strategy provides a platform for the identification of regulatory nodes and cascades that govern mesodermal and cardiovascular development.
Introduction: Duchenne Muscular Dystrophy (DMD) is the most common and debilitating X-linked muscular dystrophy characterized by absence of dystrophin. A significant cardiomyopathic phenotype is seen in DMD and has emerged as a leading cause of death in DMD. There is limited knowledge of pathophysiology of DMD cardiomyopathy due to the limited availability of patient tissues and no human cardiomyocyte model of DMD exists. We hypothesized the pathophysiology of DMD cardiomyopathy can be modeled by utilizing DMD hiPSC CM and interrogating them at the cellular and molecular level. Methods: Dermal fibroblasts were obtained from patients with DMD cardiomyopathy (dystrophin deletion of exon 4-43) and normal healthy controls. The fibroblasts were reprogrammed to hiPSC using retroviral vectors containing the human transcription factors, OCT4, SOX2, KLF4 and C-MYC. The DMD and control hiPSC lines were differentiated to CM using a directed differentiation protocol. Molecular and physiologic assays were performed at...
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.
Journal Article Reconstitution of the functional granulocyte macrophage colony stimulating factor promoter: evidence for distinct activation mechanisms that mediate the response to phorbol ester/calcium and human T cell leukemia virus type I Tax signals Get access Naoko Koyano-Nakagawa, Naoko Koyano-Nakagawa Search for other works by this author on: Oxford Academic PubMed Google Scholar Junji Nishida, Junji Nishida 2Present address: Department of Molecular Biology, Jichi Medical School3311-1 Yakushiji, Minamikawachimachi Kawachi-gun Tochigi 329-04, Japan Search for other works by this author on: Oxford Academic PubMed Google Scholar Naoko Arai, Naoko Arai 1Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California AvenuePalo Alto, CA 94304, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Ken-ichi Arai, Ken-ichi Arai Search for other works by this author on: Oxford Academic PubMed Google Scholar Takashi Yokota Takashi Yokota Correspondence to: T. Yokota Search for other works by this author on: Oxford Academic PubMed Google Scholar International Immunology, Volume 5, Issue 4, April 1993, Pages 345–352, https://doi.org/10.1093/intimm/5.4.345 Published: 01 April 1993 Article history Received: 13 November 1992 Accepted: 11 December 1992 Published: 01 April 1993
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