The initiation of the development of skin appendages (hair/feathers/scales) requires a signal from the competent dense dermis to the epidermis (Dhouailly, 1977).It is therefore essential to understand how to make a competent dermis.In recent years, a few studies have focused on the development of the dorsal dermis from the somitic dermomyotome.Our first aim in this review is to attempt to reconcile the available data on the origin of the dorsal dermis and summarize the present knowledge on the molecular mechanisms implicated in dermal lineage induction.Secondly, we open the discussion on the formation of a loose pre-dermal mesenchyme and more importantly of a dense dermis capable of participating in appendage development.To go further we draw a comparison between the chick and mouse systems to gain a new insight into how to initiate appendage morphogenesis and regulate the extent of hair/feather fields.

This chapter is mostly a review of the pioneering work of the Philippe Sengel school in Grenoble carried out in the late sixties and the seventies. The questions raised concerning the morphogenesis of feather tracts were approached by means of microsurgery on chick embryos. P. Sengel and his wife M. Kieny had the feeling that proteins synthesized by the neural tube were required for the formation of feather fields. It was my pleasure to carry on the story from the beginning. Although some clarifications concerning this morphogenesis have been contributed by my group and by a few other laboratories interested in this subject, the most important contributions to recent research have been the elucidation of the nature of the required messages, which will be explored further in other papers in this Issue.

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.

To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or of cells producing Shh led to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, triggered the formation of a feather-inducing dermis. These results show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least mimicked by Wnt-1.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Epidermal differentiation, as keratinocytes go through different layers to the skin surface, may imply a differential activation of Notch transmembrane proteins. In mouse, as recently shown in Drosophila, Notch activation by its ligands may be modulated by Fringe secreted proteins. Therefore, we cloned the mouse homolog of Radical-fng, synthesized riboprobes for Lunatic-fng, Manic-fng, and Radical-fng, and examined their expression during epidermal differentiation. Expression of all three genes is differentially activated during embryonic epidermal stratification. Manic-fng and Lunatic-fng are expressed in the basal layer, whereas Lunatic-fng is expressed in the granular layer and Radical-fng is restricted to the most differentiated nucleated layer. This expression decreases by a few days postnatally and can be reactivated by retinoic acid treatment, which triggers a new distribution of Fringe transcripts and a thickening of the granular layer. Therefore, Manic, Lunatic, and Radical Fringe by modulating the Notch pathway may play a key role in defining the different steps of keratinocyte differentiation.

Starvation is always accompanied by an increase in the ratio of AMP/ATP followed by activation of AMPK. It is one of the sensors for cellular energy status and is highly conserved across various species. Its role in the stage differentiation process of protozoan species like Giardia, Plasmodium, Trypanosome, and Toxoplasma has been reported. Since Entamoeba undergoes encystation in glucose-starved conditions; it intrigued us to investigate the existence and role of AMPK during the differentiation of trophozoites to the cyst. By employing in silico approaches, we have identified an AMPK homologue which is denominated here as EiAMPK (AMPK-like protein in Entamoeba invadens). Sequence and structural analysis indicate that EiAMPK is sequentially and structurally similar to the AMPK alpha subunit of other organisms. The recombinant form of EiAMPK was functionally active and in accordance, its activity was inhibited by an AMPK-specific inhibitor (eg. Compound C). The increased expression of EiAMPK during different stresses indicated that EiAMPK is a stress-responsive gene. To further investigate, whether EiAMPK has any role in encystation, we employed RNAi-mediated gene silencing that demonstrated its active involvement in encystation. It is known that Entamoeba maintains a flow of glucose from the glycolytic pathway to chitin synthesis for cyst wall formation during encystation. It is conceivable that EiAMPK might have a command over such glucose metabolism. As anticipated, the chitin synthesis was found greatly inhibited in both EiAMPK knockdown and Compound C treated cells, indicating that EiAMPK regulates the cyst wall chitin synthesis.

A possible relationship between the ability of Aspergillus fumigatus strains to invade tissues and genetic polymorphism was studied by random amplified polymorphic DNA (RAPD) analysis. One hundred randomly designed oligonucleotide decamers were examined with DNA of three reference strains, eight environmental isolates and 21 isolates from two distinct clinical situations: non-invasive aspergillosis (predominantly aspergilloma) and invasive aspergillosis. One primer (OPQ 6) was found to generate a reproducible amplification product that enabled distinction between the two groups according to the presence or absence of a 0.95-kb fragment that correlated with the nature of the infection (non-invasive or invasive) and immune status of the patient. The results indicated that the pathogenicity of A. fumigatus was related not only to the host's immune status but also to the virulence of the strain of A. fumigatus.

Chloroquine antimalarial action was assessed by the analysis of changes in gene expression. With this aim, Plasmodium falciparum cultures were submitted to chloroquine and to other stresses to determine which transcripts were specifically induced. P. falciparum in vitro control culture was compared to cultures where chloroquine was added and to cultures where serum was omitted, or where higher partial oxygen pressure was used, and, finally, at a temperature of 40 degrees C instead of 37 degrees C. Poly (A)(+)RNAs were reverse-transcribed and detected by the differential display technique. Two specific cDNAs were obtained and cloned, and a part of the genes was sequenced. The deduced protein, referred to as Pfhel-1, was related to a RNA helicase and was thought to be involved in protein translation control. The second deduced protein, called Pfhel-2, possessed consenses sequences of ATP-dependent helicase domains. Pfhel-2 may be involved either in mitotic control or in DNA repair. The possible roles of both helicase-related genes in chloroquine therapeutic activity are discussed.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, undergoes an asexual multiplication in man and a sexual one in mosquito. The asexual cycle can be reproduced in vitro. The present work reports the isolation of a small guanosine triphosphate-binding protein in Plasmodium falciparum extracts. This protein, a 21,000 M(r) Ras-like molecule, was revealed by western blotting in each stage of the intraerythrocytic asexual life cycle. Conversely, a 46,000 M(r) G alpha subunit of a heterotrimeric GTP-binding protein was found to be expressed during a short period from mature schizonts to free merozoites. In order to provide additional evidence for the presence of these GTP-binding proteins in Plasmodium falciparum cultures and also to determine the kinetics, we tested two toxins that are involved in the cellular signalling transduction. We observed that pertussis toxin increases P. falciparum growth, whereas cholera toxin induces crisis forms, and subsequent parasite death within the following 24 h.

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozon Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host Des sequences homologues des oncogenes des retrovirus sont retrouvees de maniere hautement conservee chez les vertebres, les insectes et les levures. Dans ce travail sept oncogenes differents ont ete utilises comme sonde pour rechercher des sequences homologues dans le genome du protozoaire Plasmodium falciparum. Les sondes v-fms et v-Ha ras s'hybrident de maniere specifique avec des sequences homologues d'ADN de P. falciparum. L'etude des oncogenes permettra de mieux connaitre la biologie de ce parasite et de comprendre notamment les relations hote-parasite qui permettent a P. falciparum de se developper en maintenant un certain niveau d'equilibre avec l'organisme humain qui l'heberge

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozon Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.

A longitudinal study was carried out in Burkina Faso to investigate the natural development of the immune response to Plasmodium falciparum malaria. Three bleedings were carried out before, during, and after the seasonal peak of transmission. Detailed antigen mapping and antibody prevalence of the 248 collected serum samples were established by immunoblotting on the basis of several epidemiological and biological parameters. An improved Western immunoblotting system was used to analyze up to 67 serum samples on each nitrocellulose sheet. This system allowed us to perform the entire study with strictly comparable conditions. Two different blood-stage antigens (exoantigens and somatic antigens) were used to analyze the distribution of different classes and subclasses of immunoglobulins according to the age of the individuals, the presence or absence of a malarial attack, the transmission period, the origin of parasite isolates, and the response to intraerythrocytic stages. Although this analysis emphasizes strong individual variations, reactions with two major antigens of 115 and 103 kDa were especially noted. These antigens induced high antibody levels and prevalences but were probably not involved in protection. The prevalence of immunoglobulin G (IgG) antibodies differed by isotype. Most of antigens stimulating IgG production were also responsible for the IgM antibody response. The role played by these antibodies in the development of natural immunity against malaria is discussed.

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.