YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Mitofusins are required for angiogenic function and modulate different signaling pathways in cultured endothelial cells.

The mitofusin proteins MFN1 and MFN2 function to maintain mitochondrial networks by binding one another and initiating outer mitochondrial membrane fusion. While it has recently been recognized that vascular endothelial cells rely upon mitochondria as signaling rather than energy-producing moieties, the role of mitochondrial dynamics in endothelial cell function has not been addressed. To begin to understand what role mitochondrial dynamics play in this context, we examined the regulation of MFN1 and MFN2 and the consequences of siRNA-mediated knockdown of these proteins in cultured endothelial cells. Treatment with VEGF-A led to the upregulation of MFN2 and, to a lesser extent, MFN1. Knockdown of either MFN led to disrupted mitochondrial networks and diminished mitochondrial membrane potential. Knockdown of either MFN decreased VEGF-mediated migration and differentiation into network structures. MFN ablation also diminished endothelial cell viability and increased apoptosis under low mitogen conditions. Knockdown of MFN2 uniquely resulted in a decrease in the generation of reactive oxygen species as well as the blunting of the gene expression of components of the respiratory chain and transcription factors associated with oxidative metabolism. In contrast, ablation of MFN1 led to the selective reduction of VEGF-stimulated Akt-eNOS signaling. Taken together, our data indicate that mitochondrial dynamics, particularly those mediated by the mitofusins, play a role in endothelial cell function and viability.

Mitofusin-2 maintains mitochondrial structure and contributes to stress-induced permeability transition in cardiac myocytes.

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outer mitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is a mediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance of mitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomorphic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrial dysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeability transition downstream of Ca(2+) stimulation or due to local generation of reactive oxygen species (ROS). Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducing stimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude that in cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells to mitochondrial permeability transition and to trigger cell death.

Adipokines in inflammation and metabolic disease.

The worldwide epidemic of obesity has brought considerable attention to research aimed at understanding the biology of adipocytes (fat cells) and the events occurring in adipose tissue (fat) and in the bodies of obese individuals. Accumulating evidence indicates that obesity causes chronic low-grade inflammation and that this contributes to systemic metabolic dysfunction that is associated with obesity-linked disorders. Adipose tissue functions as a key endocrine organ by releasing multiple bioactive substances, known as adipose-derived secreted factors or adipokines, that have pro-inflammatory or anti-inflammatory activities. Dysregulated production or secretion of these adipokines owing to adipose tissue dysfunction can contribute to the pathogenesis of obesity-linked complications. In this Review, we focus on the role of adipokines in inflammatory responses and discuss their potential as regulators of metabolic function.

Both primitive and definitive blood cells are derived from Flk-1+ mesoderm.

Emerging evidence suggests that all hematopoietic and endothelial cells originate from Flk-1(+) mesoderm in the mouse. However, this concept has not been completely proven, especially for the origin of blood cells. Using either Flk1(+/Cre);Rosa26R-EYFP or Flk1(+/Cre);Rosa26R-LacZ mice, we permanently marked Flk-1(+) cells and their progenies to determine the relationship between hematopoietic tissues and cells that express Flk-1. In embryos, all blood cells within the yolk sac and aorta were of Flk-1(+) origin. In addition, nearly all CD45(+) cells in bone marrow and circulating blood in adults were of Flk-1(+) origin. These results provide clear evidence that all blood cells, primitive and definitive, in mice are derived from Flk-1(+) mesodermal cells.

Differentiation of mouse embryonic stem cells into blood.

Embryonic stem (ES) cells can be maintained as pluripotent stem cells or induced to differentiate into many different somatic cell types. As ES-derived somatic cells can potentially be used for cell transplantation or cell-based therapy, ES cells have gained much scientific and general public attention. Successful derivation of blood from ES cells for tissue engineering will require a comprehensive understanding of inductive signals and downstream effectors involved in blood lineage development. Ideally, directed differentiation of ES cells into blood and isolation of pure hematopoietic progenitors will enhance our ability to utilize ES-derived blood cells for future clinical applications. The protocols provided in this unit describe methods of maintaining and differentiating mouse ES cells as well as identifying and isolating hematopoietic progenitors by utilizing flow cytometry and progenitor assays.

Molecular hallmarks of endogenous chromatin complexes containing master regulators of hematopoiesis.

Combinatorial interactions among trans-acting factors establish transcriptional circuits that orchestrate cellular differentiation, survival, and development. Unlike circuits instigated by individual factors, efforts to identify gene ensembles controlled by multiple factors simultaneously are in their infancy. A paradigm has emerged in which the important regulators of hematopoiesis GATA-1 and GATA-2 function combinatorially with Scl/TAL1, another key regulator of hematopoiesis. The underlying mechanism appears to involve preferential assembly of a multimeric complex on a composite DNA element containing WGATAR and E-box motifs. Based on this paradigm, one would predict that GATA-2 and Scl/TAL1 would commonly co-occupy such composite elements in cells. However, chromosome-wide analyses indicated that the vast majority of conserved composite elements were occupied by neither GATA-2 nor Scl/TAL1. Intriguingly, the highly restricted set of GATA-2-occupied composite elements had characteristic molecular hallmarks, specifically Scl/TAL1 occupancy, a specific epigenetic signature, specific neighboring cis elements, and preferential enhancer activity in GATA-2-expressing cells. Genes near the GATA-2-Scl/TAL1-occupied composite elements were regulated by GATA-2 or GATA-1, and therefore these fundamental studies on combinatorial transcriptional mechanisms were also leveraged to discover novel GATA factor-mediated cell regulatory pathways.

ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification.

FLK1-expressing (FLK1(+)) mesoderm generates blood and vessels. Here, we show that combined BMP, Notch, and Wnt signaling is necessary for efficient FLK1(+) mesoderm formation from embryonic stem cells (ESCs). Inhibition of BMP, Notch, and Wnt signaling pathways greatly decreased the generation of FLK1(+) mesoderm and expression of the Ets transcription factor Er71. Enforced expression of ER71 in ESCs resulted in a robust induction of FLK1(+) mesoderm; rescued the generation of FLK1(+) mesoderm when blocked by BMP, Notch, and Wnt inhibition; and enhanced hematopoietic and endothelial cell generation. Er71-deficient mice had greatly reduced FLK1 expression, died early in gestation, and displayed severe blood and vessel defects that are highly reminiscent of the Flk1 null mouse phenotype. Collectively, we provide compelling evidence that ER71 functions downstream of BMP, Notch, and Wnt signals and regulates FLK1(+) mesoderm, blood, and vessel development.

GATA2 functions at multiple steps in hemangioblast development and differentiation.

Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4 and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and endothelial cells.

Developmental relationship between hematopoietic and endothelial cells.

Blood (hematopoietic cells) and blood vessels (endothelial cells) develop from mesoderm via a transitional progenitor known as the hemangioblast. Flk-1, a receptor tyrosine kinase, and Scl, a basic helix-loop-helix transcription factor, are two critical molecules functioning in this process. Recent studies have shown that Flk-1 expressing mesoderm contributes to the circulatory system, including hematopoietic, endothelial, smooth muscle, skeletal muscle, and cardiac muscle cells. Our studies suggest that hemangioblast specification within Flk-1 expressing mesoderm is regulated by Scl expression. Herein, we review studies that have utilized transgenic mouse models as well as an in vitro model of embryonic stem cell differentiation, both of which have greatly contributed to the current understanding of the cellular and molecular pathways regulating hemangioblast development and differentiation.

Stepwise commitment from embryonic stem to hematopoietic and endothelial cells.

There is great excitement in generating different types of somatic cells from in vitro differentiated embryonic stem (ES) cells, because they can potentially be utilized for therapies for human diseases for which there are currently no effective treatments. Successful generation and application of ES-derived somatic cells requires better understanding of molecular mechanisms that regulate self-renewal and lineage commitment. Accordingly, many studies are aimed toward understanding mechanisms for maintaining the stem cell state and pathways leading to lineage specification. In this chapter we discuss recent studies that examine molecules that are critical for ES cell self-renewal, as well as hematopoietic and endothelial cell lineage differentiation from ES cells.

Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.

Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.