Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

TGF-ß signaling molecules in Hydra: role of BMP and BMP inhibitors during pattern formation.

Understanding the evolution of body plans has been one of the major areas of investigation in developmental and evolutionary biology. Cnidaria, the sister group to bilaterians, provides an opportunity to elucidate the origin and evolution of body axes. Hydra, a freshwater cnidarian, is a useful model to study signaling pathways governing pattern formation, which are conserved up to vertebrates including humans. The transforming growth factor ß (TGF-ß) signaling pathway is one of the fundamental pathways that regulate axis formation and organogenesis during embryonic development. In this article, we discuss the TGF-ß pathway members identified in Hydra along with other cnidarians with an emphasis on bone morphogenetic proteins (BMPs) and their inhibitors. TGF-ß members, especially those involved in BMP signaling pathway, are mainly involved in maintaining the Organizer region and patterning the body axis in Hydra. Identification of other members of this pathway in Hydra and fellow cnidarians would provide insights into the evolution of body axes and pattern formation in more complex metazoans.

Glutaredoxin 1 from Evolutionary Ancient Hydra: Characteristics of the Enzyme and Its Possible Functions in Cell.

Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

Xeroderma pigmentosum A homolog from Hydra partially complements DNA repair defect in human XPA-deficient cells.

Nucleotide excision repair (NER) pathway is a DNA repair mechanism that rectifies a wide spectrum of DNA lesions. Xeroderma pigmentosum group of proteins (XPA through XPG) orchestrate the NER pathway in humans. We have earlier studied XPA homolog from Hydra (HyXPA) and found it to be similar to human XPA. Here, we examined if HyXPA can functionally complement human XPA-deficient cells and reduce their sensitivity to UV radiation. We found that HyXPA was able to partially rescue XPA-deficient human cells from UV by its binding to chromatin of UV-irradiated cells. However, HyXPA failed to bind replication protein A (RPA70), a key interacting partner of human XPA in NER pathway. This could be attributed to changes in certain amino acid residues that have occurred during evolution, leading to prevention of some interactions between Hydra and human proteins.

DNA Repair Repertoire of the Enigmatic Hydra.

Since its discovery by Abraham Trembley in 1744, hydra has been a popular research organism. Features like spectacular regeneration capacity, peculiar tissue dynamics, continuous pattern formation, unique evolutionary position, and an apparent lack of organismal senescence make hydra an intriguing animal to study. While a large body of work has taken place, particularly in the domain of evolutionary developmental biology of hydra, in recent years, the focus has shifted to molecular mechanisms underlying various phenomena. DNA repair is a fundamental cellular process that helps to maintain integrity of the genome through multiple repair pathways found across taxa, from archaea to higher animals. DNA repair capacity and senescence are known to be closely associated, with mutations in several repair pathways leading to premature ageing phenotypes. Analysis of DNA repair in an animal like hydra could offer clues into several aspects including hydra's purported lack of organismal ageing, evolution of DNA repair systems in metazoa, and alternative functions of repair proteins. We review here the different DNA repair mechanisms known so far in hydra. Hydra genes from various DNA repair pathways show very high similarity with their vertebrate orthologues, indicating conservation at the level of sequence, structure, and function. Notably, most hydra repair genes are more similar to deuterostome counterparts than to common model invertebrates, hinting at ancient evolutionary origins of repair pathways and further highlighting the relevance of organisms like hydra as model systems. It appears that hydra has the full repertoire of DNA repair pathways, which are employed in stress as well as normal physiological conditions and may have a link with its observed lack of senescence. The close correspondence of hydra repair genes with higher vertebrates further demonstrates the need for deeper studies of various repair components, their interconnections, and functions in this early metazoan.

Differential expression of BMP inhibitors gremlin and noggin in Hydra suggests distinct roles during budding and patterning of tentacles.

BACKGROUND: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. RESULTS: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3ß inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. CONCLUSIONS: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

The past and present of developmental biology in India.

This issue of The International Journal of Developmental Biology (Int. J. Dev. Biol.) is devoted to contributions to developmental biology from India. The articles have been organized thematically, beginning with historical accounts and personal reminiscences, followed by surveys of areas to which the authors' own contributions have been substantial, and ending with reports of original research. The articles selected for the 'history' section are by those who have witnessed events from close quarters, and in most cases have contributed to the work in question. The range of articles is vast but cannot claim to be comprehensive. Some areas may have been left out inadvertently, either because we were unable to find anyone to cover them, or maybe in part because of not looking in the right place. Other areas are missed out because, much to our regret, authors did not deliver promised manuscripts on time. In short, the Special Issue is indicative of what went on and is going on in the field of developmental biology in India, but it does have gaps.

Cell signaling molecules in hydra: insights into evolutionarily ancient functions of signaling pathways.

Hydra, a Cnidarian believed to have been evolved about 60 million years ago, has been a favorite model for developmental biologists since Abraham Trembley introduced it in 1744. However, the modern renaissance in research on hydra was initiated by Alfred Gierer when he established a hydra laboratory at the Max Plank Institute in Göttingen in the late 1960s. Several signaling mechanisms that regulate development and pattern formation in vertebrates, including humans, have been found in hydra. These include Wnt, BMP, VEGF, FGF, Notch, and RTK signaling pathways. We have been using hydra to understand the evolution of cell signaling for the past several years. In this article, I will summarize the work on cell signaling pathways in hydra with emphasis on our own work. We have identified and characterized, for the first time, the hydra homologs of the BMP inhibitors Noggin and Gremlin, the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and several receptor tyrosine kinases (RTKs). Our work, along with that of others, clearly demonstrates that these pathways arose early in evolution to carry out functions that were often quite different from their functions in more complex animals. Apart from providing insights into morphogenesis and pattern formation in adult, budding and regenerating hydra, these findings bring out the utility of hydra as a model system to study evolutionarily ancient, in contrast to recently acquired, functions of various biological molecules.

Leela Mulherkar and the teaching of developmental biology.

The formal teaching of developmental biology in India began in the late nineteen-fifties at the Department of Zoology of the University of Poona. This was due to the efforts of Leela Mulherkar, who on her return from C.H. Waddington's laboratory in Edinburgh, took up the teaching of embryology at the Master's level. Mulherkar began using locally available material to teach how animals develop. They included the embryos of chicken, frog, garden lizard and molluscs, as well as organisms such as hydra and sponges. Her teaching was supported by an active research laboratory that used all these systems to address a variety of questions in embryology and teratology. She used chick embryo explants cultured in vitro extensively in her work. Teaching and research in embryology at the master's and doctoral levels at Poona University subsequently led, in 1977, to the establishment of the Indian Society of Developmental Biologists (InSDB), which is among the most active scientific societies in India.

A brief history of the Indian Society of Developmental Biologists (InSDB).

The Indian Society of Developmental Biologists (InSDB) was founded in 1977 to promote regular interactions between all those interested in how organisms develop. Conferences and training workshops are regularly held in different parts of the country. In addition to Indian developmental biologists, InSDB invites participants from different parts of the globe every year, which allows exchange of ideas with the international community. The Society, which currently has over 400 members, holds meetings every two years. For updated information, visit http://www.devbioindia.org.

One of the most significant discoveries in Developmental Biology from an Indian laboratory - Iqbal A. Niazi and the role of retinoids in limb regeneration.

In this short commentary, we reflect upon the fascinating paper by I.A. Niazi entitled "Background to work on retinoids and amphibian limb regeneration: Studies on anuran tadpoles - a retrospect." originally published in J. Biosciences (1996), and herein reproduced with the kind permission of the author and the Journal of Biosciences. It is fitting that this landmark publication is included in this India-related Special Issue of the Int. J. Dev. Biol., because it sketches the background to what is arguably one of the two most significant discoveries in Developmental Biology to come from an Indian laboratory. Besides being of intrinsic interest, it spawned an entire area of research, one that deals with the role of retinoids in morphogenesis and development generally.

Excess hydrogen peroxide inhibits head and foot regeneration in hydra by affecting DNA repair and expression of essential genes.

Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2 O2 ) is the most commonly used ROS as second messenger in the cell. It is a relatively long-lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury-induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2 O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2 O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2 O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of ß-catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2 O2 . Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.

VEGF and FGF signaling during head regeneration in hydra.

BACKGROUND: Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways play important roles in the formation of the blood vascular system and nervous system across animal phyla. We have earlier reported VEGF and FGF from Hydra vulgaris Ind-Pune, a cnidarian with a defined body axis, an organized nervous system and a remarkable ability of regeneration. We have now identified three more components of VEGF and FGF signaling pathways from hydra. These include FGF-1, FGF receptor 1 (FGFR-1) and VEGF receptor 2 (VEGFR-2) with a view to deciphering their possible roles in regeneration. METHODS: In silico analysis of proteins was performed using Clustal omega, Swiss model, MEGA 7.0, etc. Gene expression was studied by whole mount in situ hybridization. VEGF and FGF signaling was inhibited using specific pharmacological inhibitors and their effects on head regeneration were studied. RESULTS: Expression patterns of the genes indicate a possible interaction between FGF-1 and FGFR-1 and also VEGF and VEGFR-2. Upon treatment of decapitated hydra with pharmacological inhibitor of FGFR-1 or VEGFR-2 for 48 h, head regeneration was delayed in treated as compared to untreated, control regenerates. When we studied the expression of head specific genes HyBra1 and HyKs1 and tentacle specific gene HyAlx in control and treated regenerates using whole mount in situ hybridization, expression of all the three genes was found to be adversely affected in treated regenerates. CONCLUSIONS: The results suggest that VEGF and FGF signaling play important roles in regeneration of hypostome and tentacles in hydra.

Analysis of the conserved NER helicases (XPB and XPD) and UV-induced DNA damage in Hydra.

BACKGROUND: Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. METHODS: In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. RESULTS: In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500 J/m2) by 72 h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. CONCLUSIONS: Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans.

Mannose 6-phosphate-dependent lysosomal enzyme targeting in hydra: a biochemical, immunological and structural elucidation.

Mannose 6-phosphate (M6P)-dependent lysosomal enzyme targeting to endosome/lysosome complex is poorly understood among lower invertebrates. So far, only a M6P-independent lysosomal enzyme sorting protein, named LERP, has been described in Drosophila. Here, we have identified mannose 6-phosphate receptor (MPR) homologues in Hydra vulgaris, a basal Cnidarian, at genome level and further purified a cation-dependent MPR-like protein from hydra using affinity chromatography. Structural comparisons of hydra MPRs with mammalian MPRs confirm that the residues important for interacting with the M6P ligand are conserved. Based on our results, we report for the first time the occurrence of MPR-related proteins and M6P-dependent lysosomal enzyme targeting in H. vulgaris.

DNA repair enzyme APE1 from evolutionarily ancient Hydra reveals redox activity exclusively found in mammalian APE1.

Only mammalian apurinic/apyrimidinic endonuclease1 (APE1) has been reported to possess both DNA repair and redox activities. C terminal of the protein is required for base excision repair, while the redox activity resides in the N terminal due to cysteine residues at specific positions. APE1s from other organisms studied so far lack the redox activity in spite of having the N terminal domain. We find that APE1 from the Cnidarian Hydra exhibits both endonuclease and redox activities similar to mammalian APE1. We further show the presence of the three indispensable cysteines in Hydra APE1 for redox activity by site directed mutagenesis. Importance of redox domain but not the repair domain of APE1 in regeneration has been demonstrated by using domain-specific inhibitors. Our findings clearly demonstrate that the redox function of APE1 evolved very early in metazoan evolution and is not a recent acquisition in mammalian APE1 as believed so far.

Identification and characterization of the autophagy-related genes Atg12 and Atg5 in hydra.

Autophagy is an evolutionarily conserved process in eukaryotic cells that is involved in the degradation of cytoplasmic contents including organelles via the lysosome. Hydra is an early metazoan which exhibits simple tissue grade organization, a primitive nervous system, and is one of the classical non-bilaterian models extensively used in evo-devo research. Here, we describe the characterization of two core autophagy genes, Atg12 and Atg5, from hydra. In silico analyses including sequence similarity, domain analysis, and phylogenetic analysis demonstrate the conservation of these genes across eukaryotes. The predicted 3D structure of hydra Atg12 showed very little variance when compared to human Atg12 and yeast Atg12, whereas the hydra Atg5 predicted 3D structure was found to be variable, when compared with its human and yeast homologs. Strikingly, whole mount in situ hybridization showed high expression of Atg12 transcripts specifically in nematoblasts, whereas Atg5 transcripts were found to be expressed strongly in budding region and growing buds. This study may provide a framework to understand the evolution of autophagy networks in higher eukaryotes.