Protective effect of Aquaphilus dolomiae extract-G1, ADE-G1, on tight junction barrier function in a Staphylococcus aureus-infected atopic dermatitis model.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. AIMS: Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. METHODS: An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). RESULTS: Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). CONCLUSIONS: ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Schnurri mediates Dpp-dependent repression of brinker transcription.

Signalling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signalling molecules, controls many aspects of Drosophila development by activating and repressing target genes. Several essential components of the Dpp signalling pathway have been identified, including the Dpp receptors Punt and Thick veins (Tkv) as well as the cytoplasmic mediators Mad and Medea. For target genes to be activated, Dpp signalling must suppress transcription of a repressor encoded by the brinker (brk) gene. Here we show that Schnurri (Shn), a large zinc-finger protein, is essential for Dpp-mediated repression of brk transcription; in contrast, Shn is not required for target-gene activation. Thus, the Dpp signalling pathway bifurcates, downstream of the signal-mediating SMAD proteins, into a Shn-dependent pathway leading to brk repression and a Shn-independent pathway leading to gene activation. The existence of several Shn-like proteins in vertebrates and the observation that Brk functions in BMP signalling in Xenopus indicates that a similar regulatory cascade may be conserved in higher organisms.

Ubiquitous expression of sevenless: position-dependent specification of cell fate.

Specification of cell fate in the compound eye of Drosophila appears to be controlled entirely by cell interactions. The sevenless gene is required for the correct determination of one of the eight photoreceptor cells (R7) in each ommatidium. It encodes a transmembrane protein with a tyrosine kinase domain and is expressed transiently on a subpopulation of ommatidial precursor cells including the R7 precursors. It is shown here that heat shock-induced indiscriminate expression of a sevenless complementary DNA throughout development can correctly specify R7 cell identity without affecting the development of other cells. Furthermore, discontinuous supply of sevenless protein during eye development leads to the formation of mosaic eyes containing stripes of sevenless+ and sevenless- ommatidia, suggesting that R7 cell fate can be specified only within a relatively short period during ommatidial assembly. These results support the hypothesis that the specification of cell fate by position depends on the interaction of a localized signal with a receptor present on many undifferentiated cells, and that the mere presence of the receptor alone is not sufficient to specify cell fate.

Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus.

Drosophila limb development is organized by interactions between anterior and posterior compartment cells. Posterior cells continuously express and require engrailed (en) and secrete Hedgehog (Hh) protein. Anterior cells express the zinc-finger protein Cubitus interruptus (Ci). It is now shown that anterior cells lacking ci express hh and adopt posterior properties without expressing en. Increased levels of Ci can induce the expression of the Hh target gene decapentaplegic (dpp) in a Hh-independent manner. Thus, expression of Ci in anterior cells controls limb development (i) by restricting hh secretion to posterior cells and (ii) by conferring competence to respond to Hh by mediating the transduction of this signal.

Sevenless, a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain.

The determination of cell fates during the assembly of the ommatidia in the compound eye of Drosophila appears to be controlled by cell-cell interactions. In this process, the sevenless gene is essential for the development of a single type of photoreceptor cell. In the absence of proper sevenless function the cells that would normally become the R7 photoreceptors instead become nonneuronal cells. Previous morphological and genetic analysis has indicated that the product of the sevenless gene is involved in reading or interpreting the positional information that specifies this particular developmental pathway. The sevenless gene has now been isolated and characterized. The data indicate that sevenless encodes a transmembrane protein with a tyrosine kinase domain. This structural similarity between sevenless and certain hormone receptors suggests that similar mechanisms are involved in developmental decisions based on cell-cell interaction and physiological or developmental changes induced by diffusible factors.

Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal.

One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

Distinct and regulated activities of human Gli proteins in Drosophila.

In both vertebrates and Drosophila, limb development is organized by a posteriorly located source of the signalling protein Hedgehog (Hh) [1] [2] [3] [4]. In Drosophila, the expression of Hh target genes is controlled by two opposing activities of the transcriptional regulator Cubitus interruptus (Ci), which activates target genes in response to Hh signalling but is converted into a repressor form in the absence of Hh [5] [6] [7] [8] [9] [10]. Three homologs of Ci (Gli1, Gli2, and Gli3) have been implicated in mediating responses to Sonic hedgehog (Shh) in vertebrates [11] [12]. Much attention has been devoted to the expression pattern of GLI genes; GLI1 is induced by Shh, whereas GLI3 transcription appears to be repressed by Shh signalling [13] [14] [15]. The regulation of GLI gene expression is therefore one important mechanism by which GLI genes organize pattern. It is not well understood, however, whether Shh signalling also controls the activities of Gli proteins post-translationally and whether these activities have activating or repressing effects on target genes in vivo. Here, we have subjected the human proteins Gli1 and Gli3 to the precise and well-defined Hh signalling assay of Drosophila wing development and established that Gli1 functions as an activator and Gli3 as a repressor of Hh target genes; that the activating transcriptional activity of Gli1 and the repressing activity of Gli3 are both subject to Hh regulation in vivo; and that the combined activities of Gli1 and Gli3 can substitute for Ci in controlling Hh target gene expression during embryonic and larval development.

Compartment boundaries: at the edge of development.

Compartment boundaries have fascinated biologists for more than 25 years. We now know that these boundaries play important roles in pattern formation, yet how these boundaries are established during development remained a mystery. Here, we describe the exciting progress that has been made recently towards elucidating the mechanisms of boundary formation.

Hedgehog signaling in Drosophila eye and limb development - conserved machinery, divergent roles?

The secreted signaling molecule Hedgehog plays a key role in patterning Drosophila eyes and limbs. Recently, the transmembrane proteins Patched and Smoothened and the Gli protein Cubitus interruptus have been identified as essential components in Hedgehog signal transduction. Progress has also been made in understanding the function of Decapentaplegic (Dpp) in mediating the Hedgehog signal. Although playing only a minor role in the eye, Dpp governs, at long range, the expression of essential genes such as optomotor blind and spalt in the wing.

Ras1-dependent signaling by ectopically-expressed Drosophila src gene product in the embryo and developing eye.

The cellular functions of the Drosophila src 64B (Dsrc) gene product, Dsrc, and of most vertebrate Src-family kinases, are unknown. We have examined the effects of over-expression of wild type and mutated forms of Dsrc in transgenic Drosophila. Expression of both wild type Dsrc and a C-terminally truncated mutant at high levels during embryonic development induced extensive tyrosine phosphorylation of cellular proteins and caused considerable lethality, correlating with a block to germ-band retraction. Over-expression in the eye imaginal disc led to excess production of photoreceptor cells in the adult ommatidia. In contrast, expression of a kinase-inactive form of Dsrc caused distinct nervous system abnormalities in embryos and decreased the numbers of photoreceptor cells in the adult eye ommatidia. This suggests that active forms of Dsrc alter development by phosphorylation. Both the lethality and the eye roughening caused by activated Dsrc were partially suppressed by mutations in the Drosophila Ras1 gene. These results suggest that over-expressed Dsrc may function through Ras1 to stimulate differentiation in the embryonic nervous system and eye imaginal disc, and that kinase-active Dsrc interferes with these processes.

Drosophila ciD encodes a hybrid Pangolin/Cubitus interruptus protein that diverts the Wingless into the Hedgehog signaling pathway.

The Hedgehog (Hh) and Wingless (Wg) signaling pathways play important roles in animal development. The activities of the two pathways depend on each other during Drosophila embryogenesis. In the embryonic segment, Wg is required in anterior cells to sustain Hh secretion in adjacent posterior cells. Hh input in turn is necessary for anterior cells to maintain wg expression. The Hh and Wg pathways are mediated by the transcription factors Cubitus interruptus (Ci) and Pangolin/TCF (Pan), respectively. Coincidentally, pan and ci are adjacent genes on the fourth chromosome in a head-to-head orientation. Our genetic and in situ hybridization data indicate that ciD is a mutation affecting both ci and pan. Molecular analysis revealed that the ciD allele is caused by an inversion event that swapped the promoter regions and the first exons of the two genes. The ci gene in ciD is controlled by the ubiquitous pan promoter and encodes a hybrid Ci protein that carries the N-terminal region of Pan. This domain has previously been shown to bind to the b-catenin homolog Armadillo (Arm), raising the possibility that Wg input, in addition to Hh input, modulates the activity of the hybrid CiD protein. Indeed, we found that Wg signaling induces the expression of the Hh target gene patched (ptc) in ciD animals. We provide evidence that this effect depends on the ability of the CiD protein to bind Arm. Genetic and molecular data indicate that wild-type Pan and CiD compete for binding to Arm, leading to a compromised transduction of the Wg signal in heterozygous ciD/+ animals and to a dramatic enhancement of the gain-of-function activity of CiD in homozygous mutants. Thus, the Hh and the Wg pathways are affected by the ciD mutation, and the CiD fusion protein integrates the activities of both.

The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family.

Here we report the identification of Dfz3, a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signalling, but in contrast to Dfz2, it is activated rather than repressed by Wg signalling in imaginal discs. We show that Dfz3 is not required for viability but is necessary for optimal Wg signalling at the wing margin.

An ancient defense system eliminates unfit cells from developing tissues during cell competition.

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse "loser" cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development.

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model.

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. Here, we use a Drosophila tumor model to identify genes, which are specifically required for tumor growth. We found that reduction of phosphoinositide 3-kinase (PI3K) activity resulted in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. We conclude that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results presented here broaden our insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression.

Organizing activity of wingless protein in Drosophila.

The adult appendages of Drosophila are formed from imaginal discs, sheets of epithelial cells that proliferate during larval development and differentiate during metamorphosis. wingless (wg, DWnt-1) protein, a putative signaling molecule, is expressed only in prospective ventral cells in each of the leg discs. To test the role of wg, we have generated randomly positioned clones of cells that express wg protein constitutively. Clones that arise in the prospective ventral portions of the leg discs develop normally. In contrast, dorsally situated clones give rise to ventrolateral patterns and exert a ventralizing influence on neighboring wild-type tissue. We propose that wg protein organizes leg pattern along the dorsoventral axis by conferring ventral positional information within the disc.

The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic gli-binding sites.

The Drosophila Gli homolog Cubitus interruptus (Ci) controls the transcription of Hedgehog (Hh) target genes. A repressor form of Ci arises in the absence of Hh signalling by proteolytic cleavage of intact Ci, whereas an activator form of Ci is generated in response to the Hh signal. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. To investigate the mechanisms by which the two activities of Ci exert their opposite transcriptional effect, we dissect here the imaginal disc enhancer of the dpp gene, which responds to both activities of Ci. Within a minimal disc enhancer, we identify the DNA sequences that are necessary and sufficient for the control by Ci, show that the same sequences respond to the activator and repressor forms of Ci, and demonstrate that their activities can be replaced by a single synthetic Gli-binding site. We further show that the enhancer sequences of patched, a gene responding only to the activator form of Ci, effectively integrate also the repressor activity of Ci if placed into a dpp context. These results provide in vivo evidence against the employment of distinct binding sites for the different forms of Ci and suggest that target genes responding to only one form must have acquired distant cis-regulatory elements for their selective behavior.

Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein.

Drosophila limbs are subdivided into anterior and posterior compartments which derive from adjacent cell populations founded early in development. Evidence is now provided that posterior cells organize growth and cell patterning in both compartments by secreting hedgehog protein and that hedgehog protein acts indirectly by inducing neighbouring anterior cells to secrete decapentaplegic or wingless protein.

Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus.

Hedgehog (Hh) proteins play diverse organizing roles in development by regulating gene expression in responding cells. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Hh target genes. A repressor form of Ci arises in the absence of Hh signaling by proteolytic cleavage of intact Ci. We show that this cleavage is essential for limb patterning and is regulated by Hh in vivo. We provide evidence for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling.