A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

α-parvin is required for epidermal morphogenesis, hair follicle development and basal keratinocyte polarity.

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes.

Ontogenetic regulation of leukocyte recruitment in mouse yolk sac vessels.

In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.

Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem ß-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

Alpha-parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling.

During blood vessel development, vascular smooth muscle cells (vSMCs) and pericytes (PCs) are recruited to nascent vessels to stabilize them and to guide further vessel remodelling. Here, we show that loss of the focal adhesion (FA) protein alpha-parvin (alpha-pv) in mice leads to embryonic lethality due to severe cardiovascular defects. The vascular abnormalities are characterized by poor vessel remodelling, impaired coverage of endothelial tubes with vSMC/PCs and defective association of the recruited vSMC/PCs with endothelial cells (ECs). Alpha-pv-deficient vSMCs are round and hypercontractile leading either to their accumulation in the tissue or to local vessel constrictions. Because of the high contractility, alpha-pv-deficient vSMCs fail to polarize their cytoskeleton resulting in loss of persistent and directed migration. Mechanistically, the absence of alpha-pv leads to increased RhoA and Rho-kinase (ROCK)-mediated signalling, activation of myosin II and actomyosin hypercontraction in vSMCs. Our findings show that alpha-pv represents an essential adhesion checkpoint that controls RhoA/ROCK-mediated contractility in vSMCs.

Construction, database integration, and application of an Oenothera EST library.

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.