Pierre Thiebaud

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agres team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine.

Structure and Evolution of Tropomyosin Genes

Tropomyosins constitute a family of highly related actin-binding proteins found in the animal kingdom from yeast to human. In vertebrates, they are encoded by a multigene family where each member can produce several isoforms through alternative splicing and for some of them with alternate promoters. Tropomyosin isoform diversity has considerably increased during evolution from invertebrates to vertebrates and stems from the duplication of ancestral genes. The advance ofgenomic sequence information on various animals has expanded our knowledge on the structure of tropomyosin genes in different phyla and subphyla. We present the organisation of tropomyosin genes in different major phyla and the phylogenetic comparison of their structure highlights the evolution of this multigene family.

Transcription Enhancer Factor-1-dependent Expression of the α-Tropomyosin Gene in the Three Muscle Cell Types

In vertebrates, the actin-binding proteins tropomyosins are encoded by four distinct genes that are expressed in a complex pattern during development and muscle differentiation. In this study, we have characterized the transcriptional machinery of the α-tropomyosin (α-Tm) gene in muscle cells. Promoter analysis revealed that a 284-bp proximal promoter region of the Xenopus laevis α-Tm gene is sufficient for maximal activity in the three muscle cell types. The transcriptional activity of this promoter in the three muscle cell types depends on both distinct and common cis-regulatory sequences. We have identified a 30-bp conserved sequence unique to all vertebrate α-Tm genes that contains an MCAT site that is critical for expression of the gene in all muscle cell types. This site can bind transcription enhancer factor-1 (TEF-1) present in muscle cells both in vitro and in vivo. In serum-deprived differentiated smooth muscle cells, TEF-1 was redistributed to the nucleus, and this correlated with increased activity of the α-Tm promoter. Overexpression of TEF-1 mRNA in Xenopus embryonic cells led to activation of both the endogenous α-Tm gene and the exogenous 284-bp promoter. Finally, we show that, in transgenic embryos and juveniles, an intact MCAT sequence is required for correct temporal and spatial expression of the 284-bp gene promoter. This study represents the first analysis of the transcriptional regulation of the α-Tm gene in vivo and highlights a common TEF-1-dependent regulatory mechanism necessary for expression of the gene in the three muscle lineages.

Vestigial like gene family expression in Xenopus: common and divergent features with other vertebrates

The Drosophila Vestigial and Scalloped proteins form heterodimers that control wing development and are involved in muscle differentiation. Four vestigial like genes have been described in mammals. Similar to the Drosophila vestigial gene, they encode a short conserved domain (TONDU) required for interaction with the mammalian paralogues of Drosophila Scalloped (i.e., TEAD proteins). We previously identified two TEAD genes in Xenopus laevis and we report here the expression of four distinct vestigial like genes in Xenopus (vgll1-4) that represent amphibian orthologs of the mammalian vestigial like genes. Vgll1 has a unique expression pattern which is restricted to epidermal cells, both in the embryo and in the adult. Vgll2 is expressed in the skeletal muscle lineage downstream of myogenic factors and in the embryonic brain similar to the avian and mammalian orthologues. Vgll3 expression is transient, identifies embryonic hindbrain rhombomere 2, and is negatively regulated by en2, but not by egr2. Vgll4 is mainly expressed in anterior neural structures. In summary, the four Xenopus vgll genes have unique/complex expression profiles and they are differently expressed during embryogenesis. Moreover, these amphibian vestigial like genes display distinct responses to the major signaling pathways (i.e., activin, FGF or BMP) that orchestrate pattern-formation during early development.

WD Repeat-containing Protein 5, a Ubiquitously Expressed Histone Methyltransferase Adaptor Protein, Regulates Smooth Muscle Cell-selective Gene Activation through Interaction with Pituitary Homeobox 2

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.

The grape aquaporin VvSIP1 transports water across the ER membrane

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

Isolation and characterization of cDNA clones encoding the skeletal and smooth muscle Xenopus laevis β tropomyosin isoforms

Abstract cDNAs clones corresponding to the skeletal and smooth muscle β tropomyosins isoforms were isolated from a Xenopus laevis embryo cDNA library. Sequence analysis indicated that the two isoforms are coded by a single gene that uses two couples of alternative exons. The expression of the X. laevis β tropomyosin gene closely resembles that of the mammalian gene but differs from the avian gene.

Smooth muscle cell differentiation from human bone marrow: Variations in cell type specific markers and Id gene expression in a new model of cell culture

Stromal cells follow a vascular smooth muscle differentiation pathway. However, cell culture models performed from human bone marrow do not allow the obtention of a large proportion of highly differentiated smooth muscle cells (SMC) and their differentiation pathways remain unclear. We have characterized a new model of SMC differentiation from human bone marrow stromal cells by using different factors (bFGF, EGF, insulin and BMP‐4). A relative homogeneous population of differentiated SMC was reproducibly obtained in short‐term culture with high expression of SMC markers. Id gene expression was investigated and showed that (1) Id2 mRNA expression was upregulated during SMC differentiation without change of Id1 mRNA and (2) Id1 gene expression highly increased concomitantly with a decrease of SMC markers while Id2 mRNA was slightly modulated. Our data suggested that Id genes are potentially implicated in the differentiation pathway of human SMC from bone marrow.

Comparative genomic and expression analysis of the adenosine signaling pathway members in Xenopus

Adenosine is an endogenous molecule that regulates many physiological processes via the activation of four specific G-protein-coupled ADORA receptors. Extracellular adenosine may originate either from the hydrolysis of released ATP by the ectonucleotidases or from cellular exit via the equilibrative nucleoside transporters (SLC29A). Adenosine extracellular concentration is also regulated by its successive hydrolysis into uric acid by membrane-bound enzymes or by cell influx via the concentrative nucleoside transporters (SLC28A). All of these members constitute the adenosine signaling pathway and regulate adenosine functions. Although the roles of this pathway are quite well understood in adults, little is known regarding its functions during vertebrate embryogenesis. We have used Xenopus laevis as a model system to provide a comparative expression map of the different members of this pathway during vertebrate development. We report the characterization of the different enzymes, receptors, and nucleoside transporters in both X. laevis and X. tropicalis, and we demonstrate by phylogenetic analyses the high level of conservation of these members between amphibians and mammals. A thorough expression analysis of these members during development and in the adult frog reveals that each member displays distinct specific expression patterns. These data suggest potentially different developmental roles for these proteins and therefore for extracellular adenosine. In addition, we show that adenosine levels during amphibian embryogenesis are very low, confirming that they must be tightly controlled for normal development.