A disease-associated gene desert directs macrophage inflammation through ETS2.

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis3-6-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.

Xenopus fibrillin is expressed in the organizer and is the earliest component of matrix at the developing notochord-somite boundary.

We identify a Xenopus fibrillin homolog (XF), and show that its earliest developmental expression is in presumptive dorsal mesoderm at gastrulation, and that XF expression is regulated by mesoderm-inducing factors in animal cap assays. XF protein is also first detected in presumptive mesoderm, but is concentrated specifically into extracellular-matrix structures that begin to develop de novo by mid-gastrulation at both of the bilateral presumptive notochord-somite boundaries. Later in embryogenesis, XF protein is localized to the extracellular matrix at tissue boundaries, where it is found surrounding the notochord, the somites, and the neural tube, as well as under the epidermis. This pattern of protein deposition combines to give the appearance of an "embryonic skeleton," suggesting that one role for XF is to serve as a mechanical element in the embryo prior to bone deposition.

Mechanisms of convergence and extension by cell intercalation.

The cells of many embryonic tissues actively narrow in one dimension (convergence) and lengthen in the perpendicular dimension (extension). Convergence and extension are ubiquitous and important tissue movements in metazoan morphogenesis. In vertebrates, the dorsal axial and paraxial mesodermal tissues, the notochordal and somitic mesoderm, converge and extend. In amphibians as well as a number of other organisms where these movements appear, they occur by mediolateral cell intercalation, the rearrangement of cells along the mediolateral axis to produce an array that is narrower in this axis and longer in the anteroposterior axis. In amphibians, mesodermal cell intercalation is driven by bipolar, mediolaterally directed protrusive activity, which appears to exert traction on adjacent cells and pulls the cells between one another. In addition, the notochordal-somitic boundary functions in convergence and extension by 'capturing' notochordal cells as they contact the boundary, thus elongating the boundary. The prospective neural tissue also actively converges and extends parallel with the mesoderm. In contrast to the mesoderm, cell intercalation in the neural plate normally occurs by monopolar protrusive activity directed medially, towards the midline notoplate-floor-plate region. In contrast, the notoplate-floor-plate region appears to converge and extend by adhering to and being towed by or perhaps migrating on the underlying notochord. Converging and extending mesoderm stiffens by a factor of three or four and exerts up to 0.6 microN force. Therefore, active, force-producing convergent extension, the mechanism of cell intercalation, requires a mechanism to actively pull cells between one another while maintaining a tissue stiffness sufficient to push with a substantial force. Based on the evidence thus far, a cell-cell traction model of intercalation is described. The essential elements of such a morphogenic machine appear to be (i) bipolar, mediolaterally orientated or monopolar, medially directed protrusive activity; (ii) this protrusive activity results in mediolaterally orientated or medially directed traction of cells on one another; (iii) tractive protrusions are confined to the ends of the cells; (iv) a mechanically stable cell cortex over the bulk of the cell body which serves as a movable substratum for the orientated or directed cell traction. The implications of this model for cell adhesion, regulation of cell motility and cell polarity, and cell and tissue biomechanics are discussed.

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis.

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of "out-of-focus" noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers.

Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation.

DNA-protein interactions, mainly DNA-histone interactions, are thought to play an essential role in the packing mechanisms of chromatin as well as in transcriptional control. In this context it is important to develop new methods to study DNA-protein interactions and structural changes associated with them. Paraformaldehyde (PFA) has been shown to crosslink proteins, mainly histones, to DNA; and in this short study we have used a system with the DNA-binding dye 7-aminoactinomycin D (7-AAMD) as an indirect probe for PFA fixation. The aim was to investigate the dynamics of fixation on the binding of this dye to intact human lymphocytes. The results show a decrease in accessibility of 7-AAMD,initially affecting both the nonspecific binding of 7-AAMD and the high affinity binding sites, and thereafter mainly the high affinity binding sites. We conclude that fixation with PFA is a long-term reaction that requires a fixation time of several hours at a sufficient concentration to be completed. Our findings suggest that staining with a low concentration of 7-AAMD after extensive PFA fixation may be used to obtain information on DNA-protein interactions in intact cells.

High affinity binding of 7-aminoactinomycin D and 4',6-diamidino-2-phenylindole to human neutrophilic granulocytes and lymphocytes.

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction method were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid-extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.

Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos.

Xotch is a Xenopus homolog of Notch, a receptor involved in cell fate decisions in Drosophila. Using an extracellular deletion construct, Xotch delta E, we show that Xotch has a similar role in Xenopus embryos. Broad expression causes the loss of dorsal structures and the expansion and disorganization of the brain. Single blastomere injections of Xotch delta E induce autonomous neural and mesodermal hypertrophy, even in the absence of cell division. Xotch delta E inhibits the early expression of epidermal and neural crest markers yet enhances and extends the response of animal caps to mesodermal and neural induction. Our data suggest a mechanism for the function of Notch homologs in which they delay differentiation and leave undetermined cells competent to respond to later inductive signals.

XASH1, a Xenopus homolog of achaete-scute: a proneural gene in anterior regions of the vertebrate CNS.

The pro-neural achaete-scute complex (ASC) of Drosophila encodes four homologous proteins, each containing a basic helix-loop-helix (bHLH) domain, characteristic of a large family of transcription factors. We have isolated XASH1, a Xenopus homolog of achaete-scute. The XASH1 protein is very similar to the ASC proteins of Drosophila and the rat homolog, MASH1. XASH1 is expressed in the embryonic anterior central nervous system in a dynamic sequence, first in the midbrain, then in the forebrain, and then in the eye and hindbrain. In the larva, XASH1 expression correlates with regions of continued neurogenesis in the CNS, revealing the pattern of rhombomeres in the hindbrain, and other proliferative zones in the eye and midbrain. As a heterodimer with the bHLH protein E12, XASH1 binds specifically to an enhancer sequence derived from the promoter of the proneural achaete gene of Drosophila. This binding is inhibited by the extramacrochaete protein, a negative regulator of ASC gene function and neurogenesis in Drosophila. The combined evidence described in this paper strongly suggests that XASH1 plays a role in Xenopus neurogenesis similar to that played by the ASC genes in Drosophila.

Assessment of domestic violence by nurse practitioners.

Domestic violence has reached epidemic proportions in American society. In the normal course of supplying medical care to women and children, NPs will encounter victims of physical, sexual, and psychological violence. It is imperative that NPs know how to assess for victimization and safety and that they provide patients with needed information about community services.

Structural integrity of the glycoprotein IIb and IIIa genes in Glanzmann thrombasthenia patients from Israel.

Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full-length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.