Exploring the human hair follicle microbiome.

Human hair follicles (HFs) carry complex microbial communities that differ from the skin surface microbiota. This likely reflects that the HF epithelium differs from the epidermal barrier in that it provides a moist, less acidic, and relatively ultraviolet light-protected environment, part of which is immune-privileged, thus facilitating microbial survival. Here we review the current understanding of the human HF microbiome and its potential physiological and pathological functions, including in folliculitis, acne vulgaris, hidradenitis suppurativa, alopecia areata and cicatricial alopecias. While reviewing the main human HF bacteria (such as Propionibacteria, Corynebacteria, Staphylococci and Streptococci), viruses, fungi and parasites as human HF microbiome constituents, we advocate a broad view of the HF as an integral part of the human holobiont. Specifically, we explore how the human HF may manage its microbiome via the regulated production of antimicrobial peptides (such as cathelicidin, psoriasin, RNAse7 and dermcidin) by HF keratinocytes, how the microbiome may impact on cytokine and chemokine release from the HF, and examine hair growth-modulatory effects of antibiotics, and ask whether the microbiome affects hair growth in turn. We highlight major open questions and potential novel approaches to the management of hair diseases by targeting the HF microbiome.

AmAMP1 from Acropora millepora and damicornin define a family of coral-specific antimicrobial peptides related to the Shk toxins of sea anemones.

A candidate antimicrobial peptide (AmAMP1) was identified by searching the whole genome sequence of Acropora millepora for short (<125AA) cysteine-rich predicted proteins with an N-terminal signal peptide but lacking clear homologs in the SwissProt database. It resembled but was not closely related to damicornin, the only other known AMP from a coral, and was shown to be active against both Gram-negative and Gram-positive bacteria. These proteins define a family of AMPs present in corals and their close relatives, the Corallimorpharia, and are synthesised as preproproteins in which the C-terminal mature peptide contains a conserved arrangement of six cysteine residues. Consistent with the idea of a common origin for AMPs and toxins, this Cys motif is shared between the coral AMPs and the Shk neurotoxins of sea anemones. AmAMP1 is expressed at late stages of coral development, in ectodermal cells that resemble the "ganglion neurons" of Hydra, in which it has recently been demonstrated that a distinct AMP known as NDA-1 is expressed.

Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata.

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.

Control of foot differentiation in Hydra: phylogenetic footprinting indicates interaction of head, bud and foot patterning systems.

Homeodomain transcription factor CnNK-2 seems to play a major role in foot formation in Hydra. Recently, we reported in vitro evidence indicating that CnNK-2 has autoregulatory features and regulates expression of the morphogenetic peptide pedibin. We proposed that CnNK-2 and pedibin synergistically orchestrate foot differentiation processes. Here, we further analyzed the regulatory network controlling foot formation in Hydra. By phylogenetic footprinting we compared the CnNK-2 5'-flanking sequence from two closely related species, Hydra vulgaris and Hydra oligactis. Unexpectedly, we detected a highly conserved binding site for HNF-3beta, a vertebrate Forkhead transcription factor, in the CnNK-2 5'-flanking region. The Hydra HNF-3beta homolog budhead is predominantly expressed in the apical region of the body column and early during budding. Budhead is absent from tissue expressing CnNK-2 and thought to be involved in determining tissue for head differentiation. By electrophoretic mobility shift assays we demonstrate an in vitro interaction between recombinant budhead protein and the interspecific conserved HNF-3beta binding motif in the CnNK-2 5'-flanking region. Our results strengthen the view of CnNK-2 as an important regulator during foot patterning processes. Furtheron, they point to budhead as a candidate for a transcriptional regulator of CnNK-2 and to an interaction of foot and head patterning processes in Hydra on the molecular level.

Control of foot differentiation in Hydra: in vitro evidence that the NK-2 homeobox factor CnNK-2 autoregulates its own expression and uses pedibin as target gene.

The foot of the simple metazoan Hydra is a highly dynamic body region of constant tissue movement, cell proliferation, determination and differentiation. Previously, two genes have been shown to participate in the development and differentiation of this body region: homeodomain factor CnNK-2 and signal peptide pedibin [Dev. Biol. 180 (1996) 473; Development 126 (1999) 517; Development 122 (1996) 1941; Mech. Dev. 106 (2001) 37]. CnNk-2 functions as transcriptional regulator and is responsive to changes in the positional value while the secreted peptide pedibin serves as "extrinsic" positional signal. Exposure of polyps to pedibin increases the spatial domain of CnNK-2 expression towards the gastric region, indicating that positional signals are integrated at the cis-regulatory region of CnNK-2. In the present study, to elucidate the molecular basis of the interaction of CnNK-2 and pedibin, we characterized the 5' regulatory regions of both genes. Within the CnNK-2 5' upstream region, electrophoretic mobility shift assays showed that putative NK-2 binding motifs are specifically bound by both nuclear protein from Hydra foot and by recombinant CnNK-2, suggesting that CnNK-2 might autoregulate its own expression. This is the first indication for an autoregulatory circuit in Hydra. In addition, we also identified NK-2 binding sites in the cis-regulatory region of the pedibin gene, indicating that this gene is one of the targets of the transcription factor CnNK-2. On the basis of these results, we present a model for the regulatory interactions required for patterning the basal end of the single axis in Hydra which postulates that CnNK-2 together with pedibin orchestrates foot specific differentiation.

Selective protein kinase inhibitors block head-specific differentiation in hydra.

Several studies have suggested that morphogenesis and patterning in hydra are regulated through pathways involving protein kinase C (PKC). Nevertheless, the complete signal system for regeneration in hydra is still not completely understood. Using inhibitors of different signalling pathways we are dissecting this system. We found that sphingosine (2 microM), staurosporine (0.1 microM), PP1/AGL1872 (1 microM) and H7 (25 microM) were able to inhibit head but not foot regeneration. The inhibition was reversible. When the inhibitor was replaced with hydra medium the animals continue their regeneration in a normal way. The exception was PP1/AGL1872, in this case the animals regenerated only one or two tentacles. These results imply that head and foot regeneration are independent processes and they are not directly related as has been proposed. Sphingosine and PP1/AGL1872 inhibit the transcription of ks1, an early regeneration gene, at 24 and 48 h of treatment. Sphingosine 2 microM arrested the cells on the G1 phase of the cell cycle, but 1 microM of PP1/AGL1872 did not. The regeneration was not affected if the animals were exposed to inhibitors of human growth factor receptors. We propose that head regeneration in hydra may be regulated at least by two pathways, one going through PKC and the other through Src. The first pathway could be related to cellular proliferation and the second one to cellular differentiation.

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri.

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naive parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32-35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.

Cloning of a ras-related gene from Hydra which responds to head-specific signals.

Members of the Ras family of proteins are important components of signal transduction pathways responding to external signals and leading to changes in cell behavior. Analysis of two ras-related genes in the phylogenetically old metazoan Hydra indicates that in normal animals both genes are expressed in all body regions of the polyp. Upon head removal, however, the transcript level of one of the two genes, ras2, decreases rapidly in the upper gastric region which is adjacent to the former head. The decrease is transient and specific for ras2, since no changes could be observed in the transcript level of the related ras1 gene or any other gene. The disappearance of the ras2 mRNA can be prevented completely by brief exposure of decapitated polyps to the protein kinase C activator TPA, which previously was shown to be capable of converting gastric tissue into head tissue [Müller, W.A. In: Othmer, H.G. (Ed.) Experimental and Theoretical Advances in Biological Pattern Formation. Plenum Press, New York, NY, 1993, pp. 237-253]. The finding that Hydra ras2 expression is strongly dependent on a signal from the head provides the first evidence for ras expression being regulated in pattern formation.

Decision making in interstitial stem cells of Hydra.

Interstitial stem cells in Hydra are a continuously proliferating and differentiating cell population. They represent a useful model system for studying mechanisms controlling stem cell differentiation. Here we review our current knowledge of the differentiation potential of these cells. Interstitial stem cells are multipotent and able to differentiate into several different cell types. The differentiation decisions appear to be controlled by positional signals and by the composition of the cellular environment. Since interstitial stem cells can be cultured in an in vivo environment and appear to be accessible to experimental manipulation by a range of new molecular techniques, an in vivo analysis of the molecular mechanisms underlying stem cell decision making can now be approached.

Zoology www guide.

In almost every branch of zoology the internet is now a vital place for posting up-to-date status reports on all kind of animal groups and research areas. Many websites not only provide the latest on taxonomy, ecology and distribution, but also include valuable background information and details of experimental procedures as well as discussion forums. For the zoologist the online world seems to hold everything from acarology to otoliths. To show what impressive things can be done in communicating and facilitating research in zoology, given a little initiative and perseverance, we will introduce websites that find fresh ways to approach their subjects and that may, in some way or another, inspire zoologists. Readers are welcome to join in this process for future editions of Zoology www guides and should send their website suggestions to tbosch@zoologie.uni-kiel.de. In this issue the focus is on molluscs.

Zoology www guide.

As in the previous issue of Zoology, we introduce here web sites which are of interest for zoologists. Readers are welcome to make website suggestions for future editions of Zoology and should contact tbosch@zoologie.uni-kiel.de. In this issue the focus is on amphibians and reptiles.

Role of epithelial cells and programmed cell death in Hydra spermatogenesis.

Spermatogenesis in higher animals is a tightly regulated process, in which survival and death of sperm precursor cells depends on the presence of somatic cells in gonads. In the basal metazoan Hydra spermatogenesis takes place in anatomically simple testes and in the absence of accessory structures. Hydra sperm precursors are derived from interstitial stem cells. Here we show that large numbers of sperm precursors in testes of Hydra vulgaris undergo programmed cell death (apoptosis) and that ectodermal epithelial cells phagocytose the apoptotic sperm precursors. This is surprising since so far no evidence has been reported that epithelial cells are directly involved in germ cell differentiation in Hydra. We propose that, similar to Sertoli cells in mammals, in Hydra epithelial cells support and perhaps even control spermatogenesis.

Growth regulation in Hydra: relationship between epithelial cell cycle length and growth rate.

The relationship between epithelial cell production and growth rate was investigated in Hydra attenuata under different feeding regimes. The increase of epithelial cell number was compared to the duration of the epithelial cell cycle using standard methods of cell cycle analysis. The results indicate that cell cycle changes accompanying changes in feeding regime are not sufficient to explain the altered growth rate. Under heavy feeding regimes, epithelial cell production equals tissue growth rate. At low feeding level or under starvation conditions the epithelial cell cycle lengthens and growth rate of epithelial cell population is slowed. However, the cell cycle changes are insufficient to account for the reduction in tissue growth and thus there is an effective overproduction of epithelial cells amounting to 10% per day. Evidence suggests that these excess cells are phagocytized by neighboring cells in the tissue. Thus phagocytosis is directly or indirectly involved in regulating the growth of hydra tissue.

Cloned interstitial stem cells grow as contiguous patches in hydra.

The migration of interstitial cells was analyzed during the growth of stem cell clones in vivo. The spatial distribution of cloned cells was analyzed at a time by which extensive migration of interstitial cells could have occurred. All interstitial cell clones were found to form large contiguous patches of cells. The results indicate that there is little migration of large interstitial cells in undisturbed tissue during normal growth. This finding is surprising since numerous grafting experiments have shown extensive migration of these cells. The implications of finding nonrandomly distributed stem cells are discussed.

Male and female stem cells and sex reversal in Hydra polyps.

Single interstitial stem cells of male polyps of Hydra magnipapillata give rise to clones that differentiate either male or female gametes. To test the sexual stability of these clones, stem cells were recloned. The results indicate that stem cells from female clones are stable in their sexual differentiation capacity; male stem cells, by comparison, switch sexual phenotype at the rate of 10(-2) per cell per generation. As a result, female polyps contain only female stem cells; male polyps contain a mixture of male and female stem cells. A model is presented in which the sexual phenotype of Hydra polyps is controlled by (i) the switching rate of male and female stem cells and (ii) the repression of female differentiation by male stem cells.

Ontogeny recapitulates phylogeny: comparative immunology in Germany.

The innate immune system is in the spot light of modern immunology. Whenever protists, invertebrates and vertebrates are threatened by pathogens, they rapidly activate highly effective antimicrobial defense reactions. Because this young field develops very dynamically, it is important to ask what we really know about the mechanisms governing the innate immune defense system. This was the topic of a recent meeting entitled 'The Evolution of the Immune System', held at the Friedrich Schiller University in Jena, Germany. Leading scientists in the field of innate immunity presented their latest data in a historical and friendly setting.

The novel peptide HEADY specifies apical fate in a simple radially symmetric metazoan.

One of the first steps in animal development is axis formation, during which an uneven distribution of signals and/or transcription factors results in the establishment of polarity in the embryo. Hydra, one of the simplest metazoan animals, shows characteristics of a permanent embryo. Even adult polyps have a striking capacity to regenerate, suggesting that molecular mechanisms underlying de novo pattern formation are permanently active and self regulatory. Here we show that HEADY, a short, amidated peptide, plays a central role in the specification of apical fate in this simple metazoan. The HEADY gene, whose transcripts accumulate at the apical organizing center, is required for specification of apical fate, as disruption of HEADY function by dsRNA mediated interference (RNAi) results in severe defects in head formation. Conversely, an instructive role of HEADY in head specification is demonstrated by the application of synthetic HEADY peptide, which induces formation of secondary axes with head morphology. Thus, the HEADY peptide acts as developmental switch to pattern the apical-basal axis of Hydra, providing a first insight into how initial asymmetry is specified in lower metazoan animals.