Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype
Simona CHERA, Renaud de ROSA, Marijana MILJKOVIC-LICINA, Kevin DOBRETZ, Luiza GHILA, Kostas KALOULIS and Brigitte GALLIOT. J. Cell Science, 119, (in press)In hydra, the endodermal epithelial cells carry out the digestive function together with the gland cells that produce zymogens and express the orthologous gene Kazal1. In order to assess the hydra Kazal1 function, we silenced gene expression through double-stranded RNA feeding. A progressive Kazal1 silencing affected homeostatic conditions as evidenced by the low budding rate and the induced animal death. Concomitantly a dramatic disorganization followed by a massive death of gland cells was observed, while the cytoplasm of digestive cells became highly vacuolated. The presence of mitochondria and late endosomes within those vacuoles assigned them as autophagosomes. The enhanced Kazal1 expression in regenerating tips was strongly diminished in Kazal1(-) hydra, where the amputation stress led to an immediate disorganization of the gland cells, vacuolization of the digestive cells, and death after prolonged silencing. This first cellular phenotype resulting from a gene knock-down in cnidarians suggests that the Kazal1 serine protease inhibitor activity is required to prevent excessive autophagy in intact hydra and to exert a cytoprotective function to survive the amputation stress. Interestingly, these functions parallel the pancreatic autophagy phenotype observed upon mutation within the Kazal domain of the SPINK1 and SPINK3 genes in human and mice respectively.
The cAMP Response Element Binding Protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system
Simona CHERA, Kostas KALOULIS and Brigitte GALLIOT. Biosystems (in press)In biological cells, a multiplicity of extra-cellular signals can activate a unique signal transduction system that at the nuclear level will turn on a variety of target genes, eliciting thus diverse responses adapted to the initial signal. How distinct signals can converge on a unique signalling pathway that will nevertheless produce signal-specific responses provides a theoretical paradox that can be traced back early in evolution. In bilaterians, the CREB pathway connects diverse extra-cellular signals via cytoplasmic kinases to the CREB transcription factor and the CBP co-activator, regulating according to the context, cell survival, proliferation, differentiation, pro-apoptosis, long-term memory, hence achieving a « hub » function for cellular and developmental processes. In hydra, the CREB pathway is highly conserved and activated during early head regeneration through CREB phosphorylation. We show here that the CREB protein and the RSK kinase are co-expressed in all three hydra cell lineages, in dividing interstitial stem cells, in proliferating nematoblasts, spermatogonia and spermatocytes, in differentiating neurons as well as in mature ectodermal and endodermal epithelial cells. In addition, the CREB gene is up-regulated during early regeneration. When the CREB function was prevented through either exposure to the U0126 kinase inhibitor or RNA interference, head regeneration was no longer observed. Thus the CREB pathway is already involved in multiple tasks in hydra, indicating that the hub function played by the CREB pathway likely appeared very early in animal evolution. We propose that the integration of the neurogenic and patterning functions of the CREB pathway at that evolutionary time contributed to the formation of an efficient oral pole.
Regeneration in Hydra
Brigitte GALLIOT
Encyclopedia for Life Sciences,Wiley publisher. 2nd editionHydra regeneration offers a unique way to investigate ancestral molecular mechanisms leading to the establishment of organizer activity during animal development.
Relaxed constraints on Hox gene clustering during evolution
Brigitte GALLIOT. Heredity 2005. 94, 277. Pdf versionA new study of Hox genes in Oikopleura reveals these developmental genes to be scattered across the genome, rather than clustered, as found in most animals. Hox genes were initially identified in Drosophila as grouped regulatory genes, known as homeotic genes. They encode positional information during development following the colinearity rule, that is, their physical location in the cluster parallels the physical order of their expression along the anterior to posterior (AP) axis of the developing embryo (Lewis, 1978). Some years later, their molecular characterisation in both Drosophila and vertebrates proved that they code for proteins that bind DNA through the homeodomain, a domain of 60 highly evolutionarily conserved amino acids. Furthermore, mammals have the same clustered chromosomal organisation, where four copies of the Hox cluster, homologous to that of Drosophila , were found. Transcriptional analyses performed on sectioned and whole-mount embryos subsequently demonstrated the conservation of the colinearity rule (McGinnis and Krumlauf, 1992). So it seemed that Hox genes might provide a common molecular representation of the body plan at an early stage of the development of all animals. This is referred to as the phylotypic stage, during which embryos from distinct species tend to resemble to each other (Slack et al , 1993). Consequently, it was expected that the Hox gene cluster might have had this crucial developmental role even in the common ancestor of all bilaterally symmetrical animals.
The orphan COUP-TF nuclear receptors are markers for neurogenesis from cnidarians to vertebrates.
Gauchat D., Escriva H., Miljkovic-Licina M., Chera S., Langlois M-C., Begue A., Laudet V. and Galliot B.
Dev. Biol. 2004, 275, 104-123. PDF versionIn bilaterians, COUP-TF nuclear receptors participate in neurogenesis and/or CNS patterning. In hydra, the nervous system is formed of sensory mechanoreceptor cells (nematocytes) and neuronal cells, both lineages deriving from a common stem cell. The hydra COUP-TF gene, hyCOUP-TF , which encodes highly conserved DNA-binding and ligand-binding domains, belongs to the monophyletic COUP-TFs orphan receptor family (NR2F). In adult polyps, hyCOUP-TF is expressed in nematoblasts and a subset of neuronal cells. Comparative BrDU labeling analyses performed on cells expressing either hyCOUP-TF or the paired-like gene prdl-b showed that prdl-b expression corresponded to early stages of proliferation, while hyCOUP-TF was detected slightly later. HyCOUP-TF and prdl-b expressing cells disappeared in sf-1 mutants becoming b nerve-free Q . Moreover hyCOUP-TF and prdl-b expression was excluded from regions undergoing developmental processes. These data suggest that hyCOUP-TF and prdl-b belong to a genetic network that appeared together with neurogenesis during early metazoan evolution. The hyCOUP-TF protein specifically bound onto the evolutionarily conserved DR1 and DR5 response elements, and repressed transactivation induced by RAR:RXR nuclear receptors in a dose-dependent manner when expressed in mammalian cells. Hence, a cnidarian transcription factor can be active in vertebrate cells, implying that functional interactions between COUP-TF and other nuclear receptors were evolutionarily conserved.
Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system
Miljkovic-Licina M., Gauchat D. and Galliot B.
Biosystems 2004. 76, 75-87. PDF versionCnidarians represent the first animal phylum with an organized nervous system and a complex active behavior. The hydra nervous system is formed of sensory-motoneurons, ganglia neurons and mechanoreceptor cells named nematocytes, which all differentiate from a common stem cell. The neurons are organized as a nerve net and a subset of neurons participate in a more complex structure, the nerve ring that was identified in most cnidarian species at the base of the tentacles. In order to better understand the genetic control of this neuronal network, we analysed the expression of evolutionarily conserved regulatory genes in the hydra nervous system. The Prd-class homeogene prdl-b and the nuclear orphan receptor hyCOUP-TF are expressed at strong levels in proliferating nematoblasts, a lineage where they were found repressed during patterning and morphogenesis, and at low levels in distinct subsets of neurons. Interestingly, Prd-class homeobox and COUP-TF genes are also expressed during neurogenesis in bilaterians, suggesting that mechanoreceptor and neuronal cells derive from a common ancestral cell. Moreover, the Prd-class homeobox gene prdl-a , the Antp-class homeobox gene msh , and the thrombospondin-related gene TSP1 , which are expressed in distinct subset of neurons in the adult polyp, are also expressed during early budding and/or head regeneration. These data strengthen the fact that two distinct regulations, one for neurogenesis and another for patterning, already apply to these regulatory genes, a feature also identified in bilaterian related genes.
Reactivation of developmental programs: The cAMP-response element-binding protein pathway is involved in hydra head regeneration
Kaloulis, K., Chera, S., Hassel, M., Gauchat, D. and Galliot, B.
Proc. Natl. Acad. Sci., 2004. 101, 2363-2368. Pdf version; supplemental material; Fig.7 ; Fig.8Hydra regenerate throughout their life. We previously described early modulations in cAMP-response element-binding protein (CREB) DNA-binding activity during regeneration. We now show that the Ser-67 residue located in the P-box is a target for posttranslational regulation. The antihydra CREB antiserum detected CREB-positive nuclei distributed in endoderm and ectoderm, whereas the phosphoSer133-CREB antibody detected phospho-CREB-positive nuclei exclusively in endodermal cells. During early regeneration, we observed a dramatic increase in the number of phospho-CREB-positive nuclei in head-regenerating tips, exceeding 80% of the endodermal cells. We identified among CREBbinding kinases the p80 kinase, which showed an enhanced activity and a hyperphosphorylated status during head but not foot regeneration. According to biochemical and immunological evidence, this p80 kinase belongs to the Ribosomal protein S6 kinase family. Exposure to the U0126 mitogen-activated protein kinase kinase inhibitor inhibited head but not foot regeneration, abolished CREB phosphorylation and activation of the early gene HyBra1 in head-regenerating tips. These data support a role for the mitogen-activated protein kinase ribosomal protein S6 kinase CREB pathway in hydra head organizer activity.
Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra
Miljkovic, M., Mazet, F. and Galliot, B.
Dev. Biol., 2002. 246, 377-390. Pdf versionComplete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to transfect efficiently hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally when a FLAG epitope was inserted in frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken ß-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically the genetic regulations that take place in endodermal cells at the time budding or regeneration are initiated.
Cnidarians as a model system for understanding evolution and regeneration
Galliot, B and Schmid, V.
Int. J. Dev. Biol., 2002. 46, 39-48. Pdf versionHydra and Podocoryne are two cnidarian animals that provide complementary advantages for analysing developmental mechanisms possibly reflecting the basic developmental processes shared by most bilaterians. Interestingly, these mechanisms remain accessible all along the life of these animals, which bud and regenerate whatever their age. The Hydra polyp permits a direct study of the molecular cascades linking amputation to regeneration. Podocoryne displays a complete life cycle, polyp and medusa stages with a fast and inducible sexual cycle and an unparalleled in vitro transdifferentiation potential. In both cases, a large number of evolutionarily conserved molecular markers are available, and the analysis of their regulation highlight the molecular mechanisms that underly pattern formation in these two species.
Regeneration in Hydra
Galliot, B
Encyclopedia for Life Sciences,Macmillan Reference Ltd.Hydra regeneration offers a unique way to investigate ancestral molecular mechanisms leading to the establishment of organizer activity during animal development.
Conserved and Divergent Genes in Apex and Axis Development of Cnidarians.
Galliot, B.
Curr. Op. Genet. Dev.,2000, 10, 629-637. Pdf version
Despite their radial organization and their sister group position in the life tree, cnidarian species express during morphogenesis a large number of genes that are related to bilaterian developmental genes. Among those, homologs to forkhead, emx, aristaless, goosecoid, brachyury, wnt and nanos genes are regulated during apical patterning in cnidarians, suggesting that key components of early organizer activity were conserved across evolution and recruited for either anterior, axial, or dorso-ventral patterning in bilaterians. In contrast, the expression patterns of the cnidarian Hox-related genes suggest that the apical-basal axis of the cnidarian polyp and the anterior-posterior axis of bilaterians do not differentiate following homologous processes.Evolution of Antp-class Genes and Differential Expression of Hydra Hox/paraHox genes in Anterior Patterning
Gauchat, D., Mazet, F., Berney, C., Schummer, M., Kreger, S., Pawlowski, J. and Galliot, B.
Proc. Natl. Acad. Sci., 2000, 97, 4493-4498. Pdf version; Supplementary fileThe conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs, Msx, Emx, Barx, Evx, Tlx, NK-2 and the newly reported here Prh/Hex, Not and Dlx. Among the Hox/paraHox, poriferan sequences were not found and the cnidarian sequences formed at least 5 distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, while three display some relatedness to the Hox paralog groups 1 (cnox-1), 9/10 (cnox-3) and the paraHox cdx (cnox-4). Intermediate Hox/paraHox genes (PG 3 to 8 and lox) did not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2 and cnox-3 were not found chromosomally linked within a 150 kb range, and displayed specific expression patterns in the adult head. During regeneration, cnox-1 was expressed as an early gene whatever the polarity, whereas cnox-2 was up-regulated later during head but not foot regeneration. Finally, cnox-3 expression was reestablished in the adult head once it is fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However the positional information defining the oral/aboral axis in Hydra cannot strictly be correlated to that characterizing the anterior-posterior axis in vertebrates or arthropods.
How Old is Our Head ? Origin of Anterior Patterning
Galliot, B. and Miller, D.
Trends Genet., 2000, 16, 1-5. Pdf versionMost animals, which display a bilateral symmetry (bilaterians), share homologous regulatory genes involved in head development. Recently, homologues of several of those have been cloned from animals radially organised such as coral, sea anemones, jellyfish or hydra (cnidarians). Surprisingly, some of these are expressed apically and/or during apical patterning in hydrozoans, suggesting that head patterning is much older than previously thought.
Evolution of Homeobox Genes: Q50 Paired-like Genes Founded the Paired-Class
Galliot, B., de Vargas, C. and Miller, D.
Dev. Genes Evol.,1999, 209, 186-197. Pdf version
The genes belonging to the Paired class exert primary developmental functions. They are characterized by six invariant amino acid residues in the homeodomain, while the residue at position 50 can be a serine, glutamine or lysine as in the Pax-type, Q50 Paired-like or the K50 Paired-like homeodomains respectively. Genes in this class emerged early in animal evolution: three distinct Pax genes and two Q50 Paired- like genes have recently been characterised from cnidarians. Phylogenetic molecular reconstructions taking into account homeodomain and paired-domain sequences provide some new perspectives on the evolution of the Paired-class genes. Analysis of 146 Paired-class homeodomains from a wide range of metazoan taxa allowed us to identify 18 families among the three sub-classes from which the aristaless family displays the least diverged position. Both Pax-type and K50 families branch within the Q50 Paired-like sequences implying that these are the most ancestral. Consequently, most Pax genes arose from a Paired-like ancestor, via fusion of a Paired-like homebox gene with a gene encoding only a paired domain; the Cnidaria appear to contain genes representing the 'before' and 'after' fusion events.
Prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes
Gauchat, D., Kreger, S., Holstein, T. and Galliot, B.
Development, 1998. 125, 1637-1645. Pdf versionTwo homeobox genes, prdl-a and prdl-b, which were isolated from a Hydra vulgaris cDNA library, encode paired-like class homeodomains highly related to that of the aristaless-related genes. In adult polyps, prdl- b is a marker for synchronously dividing nematoblasts while prdl-a displays an expression restricted to the the nerve cell lineage of the head region. During budding and apical regeneration, an early and transient prdl-a expression was observed in endodermal cells of the stump at a time when the head organizer is established. When apical regeneration was delayed upon concomittant budding, prdl-a expression was found to be altered in the stump. Furthermore, a specific anti-prdl- a protein immunoserum revealed that prdl-a was overexpressed in adult polyps of the Chlorohydra viridissima multiheaded mutant, with an expression domain extending below the tentacle ring towards the body column. Accordingly, prdl-a DNA-binding activity was enhanced in nuclear extracts from this mutant. These results suggest that prdl-a responds to apical forming signals and might thus be involved in apical specification. When a marine hydrozoan (Podocorynae carnea) was used, the anti-prdl-a antibody showed cross-reactivity with cells located around the oral region, indicating that prdl-a function is shared by other cnidaria. The ancestral role for prdl-a-related genes in the molecular definition of the head (or oral-surrounding region) is discussed.
Signaling molecules in Hydra regeneration
Galliot, B.
BioEssays, 1997. 19, 37-46.Ever since Trembley discovered regeneration in hydra, 250 years ago(1), regeneration has remained a stimulating question for developmental biologists. During the last 25 years, cellular approaches have revealed that, within the first few hours of apical or basal hydra regeneration, differentiation and determination of nerve cells are the primary cellular events detectable(2-4). The neuropeptides head and foot activators (HA, FA), which are released upon injury, are signaling molecules involved in these processes(4-6). Recent results indicate that, in conditions where HA induces cellular differentiation or determination, HA behaves as an agonist of the cAMP pathway involving the modulation of the CREB transcription factor activity(7, 8). This cascade would be required for the proper regeneration, regardless of the polarity involved, apical or basal. Modulations of the PKC pathway which have been shown to modify apical or basal positional values, might signal to bring this polarity(9-14); however, endogenous ligands responsible for this modulation are as yet unknown.
The cAMP Response Element Binding protein is involved in hydra regeneration
Galliot, B., Welschof, M., Schuckert, O., Hoffmeister, S., and Schaller H.C.
Development, 1995. 121, 1205-1216. Pdf versionHydra provides an interesting developmental model system where pattern formation processes are easily accessible to experimentation during regeneration. Previous studies have shown that the neuropeptide head activator affects cellular growth and head-specific cellular differentiation during head regeneration and budding. In order to investigate the signal transduction pathway and the regulatory genes involved in these processes, we measured cAMP levels after head activator treatment and found that head activator leads to an increase in cAMP levels at concentrations where effects on nerve cell determination and differentiation are observed (10(-11) to 10(-9) M). Moreover, exposure of intact hydra to a permeable form of cAMP stimulates nerve-cell differentiation and thus mimicks the effect of endogenous head activator. Band-shift assays were performed to detect changes in hydra nuclear protein binding activity during regeneration or after head activator treatment. We found that the cAMP response element (CRE) promotes a specific and strong DNA-binding activity which is dramatically enhanced and modified during early regeneration or after HA treatment. We also identified a surprisingly highly conserved hydra gene encoding the cAMP Response Element Binding protein, which is involved in this CRE-binding activity. Initiation of regeneration upon wounding provokes an endogenous release of HA which leads to the final differentiation of determined nerve cells. We propose that the nerve- cell differentiation observed within the first 4-8 hours of regeneration relies on the agonist effect of head activator on the cAMP pathway, which would in turn modulate the CRE-binding activity of the hydra CREB protein and thus regulate the transcriptional activity of genes involved in regeneration processes.
The role of the cAMP pathway in mediating the effect of head activator on nerve-cell determination and differentiation in hydra
Fenger, U., Hofman, M., Galliot, B. and Schaller, H.C. Mech. Dev. 1994. 47, 115-125.In hydra, head activator (HA) acts as positive signal for nerve-cell determination and differentiation. For both events, HA uses cAMP as the second messenger. Evidence is presented that the cAMP agonist, Sp- cAMPS, is able to mimick the effect of HA on nerve-cell determination and differentiation and that it is blocked by the antagonist Rp-cAMP. An adenylyl cyclase associated protein, CAP, appears to be involved as mediator for transducing the signal from the transmembrane HA receptor to the cAMP system. A cDNA coding for hydra CAP was isolated from the multiheaded mutant of Chlorohydra viridissima. The hydra CAP shows extensive homology with the yeast and, more so, mammalian CAPs. In hydra, CAP mRNA is expressed abundantly in interstitial and epithelial cells. The effect of HA, but not of cAMP, on nerve-cell differentiation was inhibited by pretreatment of hydra with a cap antisense oligonucleotide, suggesting a role for CAP as mediator in the signal transduction cascade between HA and cAMP.
"Guessmer" screening strategy applied to species with A/T rich coding sequences
Galliot, B. and Schummer, M. Trends Genet. 1993. 9, 3-4.
HOM/HOX homeobox genes are present in hydra (Chlorohydra viridissima) and are differentially expressed during regeneration
Schummer, M., Scheurlen, I., Schaller, C. and Galliot, B. EMBO.J.,1992. 11, 1815-1823.Hydra, a diblastic animal consisting of two cell layers, ectoderm and endoderm, is one of the most ancient animals displaying an anteroposterior axis with a head and a foot developing from an uncommitted gastric region. As such, hydra is an interesting model for studying the presence and function of homeobox genes in a phylogenetically old organism. By screening a Chlorohydra viridissima cDNA library with a 'guessmer' oligonucleotide, we have cloned several such cnidarian homeobox-containing genes (cnox genes). Two of these, cnox1 and cnox2, display labial and Deformed type homeodomains respectively and could represent two ancestral genes of the HOM/HOX complexes; cnox3 exhibits some similarity to the BarH1 and the distal- less type homeodomains and a fourth gene is highly related to the msh/Hox7 type of homeodomain. We used quantitative PCR to study levels of expression of these genes along the body axis and during head regeneration. In all cases, the expression in heads was stronger than that in the gastric region. cnox1 transcripts dramatically peaked within the first hours of head regeneration, whereas cnox2 and cnox3 reached their maximal levels 1 and 2 days after cutting respectively. This differential expression of homeobox genes at various stages of regeneration suggests that they play specific roles in regenerative processes.