Dll4 Blockade Promotes Angiogenesis in Nonhealing Wounds of Sox7-Deficient Mice.

Objective: This study aimed to elucidate the role of the proangiogenic transcription factors Sox7 and Sox17 in the wound healing process and investigate the therapeutic potential of Dll4 blockade, which is an upstream regulator of Sox17, for the treatment of nonhealing wounds. Approach: After generating a full-thickness skin defect wound model of endothelial Sox7- and/or Sox17-deficient mice, we measured the wound healing rates and performed histological analysis. The effects of an anti-Dll4 antibody on wound angiogenesis in Sox7-deficient mice and db/db diabetic mice were assessed. Results: Sox7 and/or Sox17 deletion delayed wound healing. Moreover, the loss of Sox7 and Sox17 inhibited wound angiogenesis, without affecting the expression of the other. Of interest, after anti-Dll4 antibody treatment, Sox17 levels were increased and the suppression of angiogenesis was alleviated in Sox7-deficient mice and db/db diabetic mice. Consequently, Dll4 blockade effectively recovered the observed delay in wound healing. Innovation: The proangiogenic role of Sox7 and Sox17 in wound angiogenesis was addressed and effective treatment of nonhealing wounds by Dll4 blockade was suggested. Conclusion: This study revealed the proangiogenic role of the transcription factors Sox7 and Sox17 in wound angiogenesis. Furthermore, we suggest a novel method for treating nonhealing wounds by particularly targeting the Dll4-Sox17 axis.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

The SWI/SNF Protein PBRM1 Restrains VHL-Loss-Driven Clear Cell Renal Cell Carcinoma.

PBRM1 is the second most commonly mutated gene after VHL in clear cell renal cell carcinoma (ccRCC). However, the biological consequences of PBRM1 mutations for kidney tumorigenesis are unknown. Here, we find that kidney-specific deletion of Vhl and Pbrm1, but not either gene alone, results in bilateral, multifocal, transplantable clear cell kidney cancers. PBRM1 loss amplified the transcriptional outputs of HIF1 and STAT3 incurred by Vhl deficiency. Analysis of mouse and human ccRCC revealed convergence on mTOR activation, representing the third driver event after genetic inactivation of VHL and PBRM1. Our study reports a physiological preclinical ccRCC mouse model that recapitulates somatic mutations in human ccRCC and provides mechanistic and therapeutic insights into PBRM1 mutated subtypes of human ccRCC.

Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function.

The importance of canonical Wnt signaling to murine uterine development is well established. Mouse models in which uterine-specific Wnt ligands, ß-catenin, or Lef1 are disrupted result in failure of postnatal endometrial gland development. Sox17 is a transcription factor characterized in numerous tissues as an antagonist of Wnt signaling. Thus, we hypothesized that conditional ablation of Sox17 would lead to hyperproliferation of endometrial glands in mice. Contrary to our prediction, disruption of Sox17 in epithelial and stromal compartments led to inhibition of endometrial adenogenesis and a loss of reproductive capacity. Epithelium-specific Sox17 disruption resulted in normal adenogenesis although reproductive capacity remained impaired. These findings suggest that non-epithelial, Sox17-positive cells are necessary for adenogenesis and that glands require Sox17 to properly function. To our knowledge, these findings are the first to implicate Sox17 in endometrial gland formation and reproductive success. The data presented herein underscore the importance of studying Sox17 in uterine homeostasis and function.

Sox7, Sox17, and Sox18 Cooperatively Regulate Vascular Development in the Mouse Retina.

Vascular development and maintenance are controlled by a complex transcriptional program, which integrates both extracellular and intracellular signals in endothelial cells. Here we study the roles of three closely related SoxF family transcription factors-Sox7, Sox17, and Sox18 -in the developing and mature mouse vasculature using targeted gene deletion on a mixed C57/129/CD1 genetic background. In the retinal vasculature, each SoxF gene exhibits a distinctive pattern of expression in different classes of blood vessels. On a mixed genetic background, vascular endothelial-specific deletion of individual SoxF genes has little or no effect on vascular architecture or differentiation, a result that can be explained by overlapping function and by reciprocal regulation of gene expression between Sox7 and Sox17. By contrast, combined deletion of Sox7, Sox17, and Sox18 at the onset of retinal angiogenesis leads to a dense capillary plexus with a nearly complete loss of radial arteries and veins, whereas the presence of a single Sox17 allele largely restores arterial identity, as determined by vascular smooth muscle cell coverage. In the developing retina, expression of all three SoxF genes is reduced in the absence of Norrin/Frizzled4-mediated canonical Wnt signaling, but SoxF gene expression is unaffected by reduced VEGF signaling in response to deletion of Neuropilin1 (Npn1). In adulthood, Sox7, Sox17, and Sox18 act in a largely redundant manner to maintain blood vessel function, as adult onset vascular endothelial-specific deletion of all three SoxF genes leads to massive edema despite nearly normal vascular architecture. These data reveal critical and partially redundant roles for Sox7, Sox17 and Sox18 in vascular growth, differentiation, and maintenance.

Deficiency of endothelium-specific transcription factor Sox17 induces intracranial aneurysm.

BACKGROUND: Intracranial aneurysm (IA) is a common vascular disorder that frequently leads to fatal vascular rupture. Although various acquired risk factors associated with IA have been identified, the hereditary basis of IA remains poorly understood. As a result, genetically modified animals accurately modeling IA and related pathogenesis have been lacking, and subsequent drug development has been delayed. METHODS AND RESULTS: The transcription factor Sox17 is robustly expressed in endothelial cells of normal intracerebral arteries. The combination of Sox17 deficiency and angiotensin II infusion in mice induces vascular abnormalities closely resembling the cardinal features of IA such as luminal dilation, wall thinning, tortuosity, and subarachnoid hemorrhages. This combination impairs junctional assembly, cell-matrix adhesion, regeneration capacity, and paracrine secretion in endothelial cells of intracerebral arteries, highlighting key endothelial dysfunctions that lead to IA pathogenesis. Moreover, human IA samples showed reduced Sox17 expression and impaired endothelial integrity, further strengthening the applicability of this animal model to clinical settings. CONCLUSIONS: Our findings demonstrate that Sox17 deficiency in mouse can induce IA under hypertensive conditions, suggesting Sox17 deficiency as a potential genetic factor for IA formation. The Sox17-deficient mouse model provides a novel platform to develop therapeutics for incurable IA.

Wnt/ß-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse.

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse ß-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of ß-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for ß-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of ß-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/ß-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/ß-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Sox17-dependent gene expression and early heart and gut development in Sox17-deficient mouse embryos.

Sox17 is a transcription factor that is required for maintenance of the definitive endoderm in mouse embryos. By expression profiling of wild-type and mutant embryos and Sox17-overexpressing hepatoma cells, we identified genes with Sox17-dependent expression. Among the genes that were up-regulated in Sox17-null embryos and down-regulated by Sox17 expressing HepG2 cells is a set of genes that are expressed in the developing liver, suggesting that one function of Sox17 is the repression of liver gene expression, which is compatible with a role for Sox17 in maintaining the definitive endoderm in a progenitor state. Consistent with these findings, Sox17(-/-) cells display a diminished capacity to contribute to the definitive endoderm when transplanted into wild-type hosts. Analysis of gene ontology further revealed that many genes related to heart development were downregulated in Sox17-null embryos. This is associated with the defective development of the heart in the mutant embryos, which is accompanied by localised loss of Myocd-expressing cardiogenic progenitors and the malformation of the anterior intestinal portal.

Ancient news: HMGBs are universal sentinels.

Yanai et al. (2009, Nature 462, 99-103) have shown that high mobility group boxs (HMGBs) are universal sensors of viral nucleic acids, and thus of cell infection. This appears to be an evolutionary ancient mechanism of virus detection, and possibly might be a facet of a more general propensity of HMGBs to act as integrators of signals that pertain to peace and stress, life and death.

HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

Crosstalk between AHR and Wnt signaling through R-Spondin1 impairs tissue regeneration in zebrafish.

Exposure to dioxins, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a wide array of toxicities in vertebrates, which are mostly considered to be mediated through the inappropriate activation of the aryl hydrocarbon receptor (AHR) signaling pathway. Although transcriptional regulation by AHR is widely studied, the molecular mechanisms responsible for the adverse outcomes after AHR activation are largely unknown. To identify the important downstream events of AHR activation, we employed the zebrafish caudal fin regeneration model, where AHR activation blocks the regenerative process. Comparative toxicogenomic analysis revealed that both adult and larval fins respond to TCDD during regeneration with misexpression of Wnt signaling pathway members and Wnt target genes. R-Spondin1, a novel ligand for the Wnt coreceptor, was highly induced, and we hypothesized that misexpression of R-Spondin1 is necessary for AHR activation to block regeneration. Partial antisense repression of R-Spondin1 reversed the inhibitory effect of TCDD, and tissue regeneration was restored. This finding demonstrates that inhibition of regeneration by TCDD is mediated by misinduction of R-Spondin1. Because R-Spondin1 signals through the Wnt coreceptor LRP6, we further demonstrated that the TCDD-mediated block in regeneration is also LRP6 dependent. Collectively, these results indicate that inappropriate regulation of R-Spondin/LRP6 is absolutely required for TCDD to inhibit fin regeneration.

Fetal to adult stem cell transition: knocking Sox17 off.

What controls the inherent differences between fetal and adult hematopoietic stem cells (HSCs)? In this issue of Cell, Kim et al. (2007) demonstrate in mice that the endodermal transcription factor Sox17 is required for the maintenance of fetal and neonatal but not adult HSCs.

Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells.

Fetal stem cells differ phenotypically and functionally from adult stem cells in diverse tissues. However, little is known about how these differences are regulated. To address this we compared the gene expression profiles of fetal versus adult hematopoietic stem cells (HSCs) and discovered that the Sox17 transcriptional regulator is specifically expressed in fetal and neonatal but not adult HSCs. Germline deletion of Sox17 led to severe fetal hematopoietic defects, including a lack of detectable definitive HSCs. Conditional deletion of Sox17 from hematopoietic cells led to the loss of fetal and neonatal but not adult HSCs. HSCs stopped expressing Sox17 approximately 4 weeks after birth. During this transition, loss of Sox17 expression correlated with slower proliferation and the acquisition of an adult phenotype by individual HSCs. Sox17 is thus required for the maintenance of fetal and neonatal HSCs and distinguishes their transcriptional regulation from adult HSCs.

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos.

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.