Invertebrate Animal Models Research Articles

Mitochondrial Cell Death Pathways in Caenorhabiditis elegans.

Programmed cell death is an evolutionarily conserved process essential for animal development and tissue homeostasis. Mitochondria have been demonstrated to play a central role in regulating both the activation and the execution of apoptosis. In particular, mitochondria release multiple proapoptotic factors from its intermembrane space, leading to both caspase-dependent and -independent cell death. Despite the pivotal roles of invertebrate animal models, Caenorhabiditis elegans and Drosophila melanogaster, in deciphering conserved pathways and mechanisms of programmed cell death, the importance of mitochondria to apoptosis of invertebrates remains elusive and largely unexplored. Recent studies have corroborated significant association between mitochondria and apoptosis in C. elegans, making it a thrust area of investigations. In this review, we detail the roles of mitochondrial proteins in mediating execution of cell death in C. elegans, including chromosome fragmentation, phosphatidylserine externalization, and elimination of mitochondria, and discuss the potential roles of mitochondria in the activation of C. elegans cell death. The combination of traditional powerful genetic tools and the emergence of the multiple new reverse genetic techniques, including the highly efficient CRISPR/Cas9 gene-editing method, should make C. elegans an ideal animal model for analyzing mitochondrial cell death pathways and associated regulatory mechanisms.

TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour.

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca2+ influx, in most animal species. However, the relationship between ion permeation and animals’ nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca2+ influx. Within the selectivity filter, M601-F609, Y604G strongly reduces avoidance behaviour and eliminates Ca2+ transients. Y604F also abolishes Ca2+ transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y604 may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.

Longevity pathways and memory aging.

The aging process has been associated with numerous pathologies at the cellular, tissue, and organ level. Decline or loss of brain functions, including learning and memory, is one of the most devastating and feared aspects of aging. Learning and memory are fundamental processes by which animals adjust to environmental changes, evaluate various sensory signals based on context and experience, and make decisions to generate adaptive behaviors. Age-related memory impairment is an important phenotype of brain aging. Understanding the molecular mechanisms underlying age-related memory impairment is crucial for the development of therapeutic strategies that may eventually lead to the development of drugs to combat memory loss. Studies in invertebrate animal models have taught us much about the physiology of aging and its effects on learning and memory. In this review we survey recent progress relevant to conserved molecular pathways implicated in both aging and memory formation and consolidation.

Gut microbiota-generated metabolites in animal health and disease.

Gut microbiota is found in virtually any metazoan, from invertebrates to vertebrates. It has long been believed that gut microbiota, more specifically, the activity of the microbiome and its metabolic products, directly influence a variety of aspects in metazoan physiology. However, the exact molecular relationship among microbe-derived gut metabolites, host signaling pathways, and host physiology remains to be elucidated. Here we review recent discoveries regarding the molecular links between gut metabolites and host physiology in different invertebrate and vertebrate animal models. We describe the different roles of gut microbiome activity and their metabolites in regulating distinct host physiology and the molecular mechanisms by which gut metabolites cause physiological homeostasis via regulating specific host signaling pathways. Future studies in this direction using different animal models will provide the key concepts to understanding the evolutionarily conserved chemical dialogues between gut microbiota and metazoan cells and also human diseases associated with gut microbiota and metabolites.

Special invertebrate models and integrative medical applications: regulations, mechanisms, and therapies.

This special issue of eCAM has brought together several unique investigators interested in invertebrate animal models. Investigators would benefit by examining advances and applications of findings derived from useful terrestrial and marine invertebrate animal models. Results derived from well-defined evidence-based approaches are usually relevant to humans; the advantages are numerous, inexpensive, and noncontroversial and are not subject to rigid, ethical, or moral guidelines. In the historical context, these animals have been crucial in many cultures before the advent of industrial medicines thus providing the only source of aid. Now, there is a move to return to or at least to include these practices lest they disappear since, in certain cultures, they represent the sole source of useful health remedies. However, evidence-based medicine emphasizes the importance of subjecting animal products to rigorous analysis. Results are then standardized and serve to educate populations including those that use exclusively western practices. eCAM stands ready to accept the importance and contributions of bees as valuable sources of beneficial activities (pollination) and products (honey and propolis) that for centuries have enhanced human lives as food and medicine. Others include corals, millipedes, maggots, and earthworms. Corals are marine invertebrates that live in compact colonies. Humans have learned that they are useful in the manufacture of jewelry and chemical compounds that are useful against cancer, AIDS, and pain and serve as skeletons during bone grafting. Millipedes are arthropods (the same group as shrimps, crabs, butterflies, and bees) which in some cultures are used during pregnancy and as cures for fever, wounds, earaches, and hemorrhoids and food among the Bobo people of Burkina Faso when crushed; sometimes they are boiled and eaten in tomato sauce. Since the time of Napoleon and even centuries earlier, maggots have been used especially in patients with severe intractable wounds caused by nerve degeneration that results from diabetes. In fact, leeches and maggots are now valuable components of the emerging field of biotherapy, the therapeutic use of living creatures to clean gangrenous tissue often found in ulcers, burns, and postoperative infections. Earthworms are experiencing growing popularity due to their many applications including sources of high protein as food and the production of an anticlotting agent known as lumbrokinase that dissolves blood clots in patients. Aside from observations and usefulness by the layman, certain techniques are valuable as molecular biologists strive to understand the minute structure of molecules in order to improve therapies. Let us not forget the contributions of earthworms as tillers of the soil which enhance agricultural output. Earthworms keep the soil aerated by their constant churning which makes for improved yield of plants regardless of the proposed use. Chih-Yang Huang Edwin L. Cooper Catherine Fang-Yeu Poh Wei-Wen Kuo Tung-Sheng Chen Ronald Sherman

A fucose containing polymer-rich fraction from the brown alga Ascophyllum nodosum mediates lifespan increase and thermal-tolerance in Caenorhabditis elegans, by differential effects on gene and protein expression.

The extracts of the brown alga, Ascophyllum nodosum, which contains several bioactive compounds, have been shown to impart biotic and abiotic stress tolerance properties when consumed by animals. However, the physiological, biochemical and molecular mechanism underlying such effects remain elusive. We investigated the effect of A. nodosum fucose-containing polymer (FCP) on tolerance to thermally induced stress using the invertebrate animal model, Caenorhabditis elegans. FCP at a concentration of 150 μg mL(-1) significantly improved the life span and tolerance against thermally induced stress in C. elegans. The treatment increased the C. elegans survival by approximately 24%, when the animals were under severe thermally induced stress (i.e. 35 °C) and 27% under mild stress (i.e. 30 °C) conditions. The FCP induced differential expression of genes and proteins is associated with stress response pathways. Under thermal stress, FCP treatment significantly altered the expression of 65 proteins (54 up-regulated & 11 down-regulated). Putative functional analysis of FCP-induced differential proteins signified an association of altered proteins in stress-related molecular and biochemical pathways of the model worm.

The chick embryo as an expanding experimental model for cancer and cardiovascular research

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.

Effect of Nanoparticles on the Biochemical and Behavioral Aging Phenotype of the NematodeCaenorhabditis elegans

Invertebrate animal models such as the nematode Caenorhabditis elegans (C. elegans) are increasingly used in nanotechnological applications. Research in this area covers a wide range from remote control of worm behavior by nanoparticles (NPs) to evaluation of organismal nanomaterial safety. Despite of the broad spectrum of investigated NP-bio interactions, little is known about the role of nanomaterials with respect to aging processes in C. elegans. We trace NPs in single cells of adult C. elegans and correlate particle distribution with the worm's metabolism and organ function. By confocal microscopy analysis of fluorescently labeled NPs in living worms, we identify two entry portals for the uptake of nanomaterials via the pharynx to the intestinal system and via the vulva to the reproductive system. NPs are localized throughout the cytoplasm and the cell nucleus in single intestinal, and vulval B and D cells. Silica NPs induce an untimely accumulation of insoluble ubiquitinated proteins, nuclear amyloid and reduction of pharyngeal pumping that taken together constitute a premature aging phenotype of C. elegans on the molecular and behavioral level, respectively. Screening of different nanomaterials for their effects on protein solubility shows that polystyrene or silver NPs do not induce accumulation of ubiquitinated proteins suggesting that alteration of protein homeostasis is a unique property of silica NPs. The nematode C. elegans represents an excellent model to investigate the effect of different types of nanomaterials on aging at the molecule, cell, and whole organism level.

Hematology from the Ground Up: Lessons from C. elegans

Heme is a vital cofactor responsible for diverse biological functions such as gas synthesis and sensing, circadian clock control, microRNA processing, and xenobiotic detoxification. Heme synthesis occurs in the mitochondria in eukaryotes. Free heme is a hydrophobic and cytotoxic macrocycle. How then is heme transported through cellular membranes and organelles? What are the mechanisms for incorporating heme into specific hemoproteins that reside in the cytoplasm, peroxisomes, mitochondria, secretory pathway, and nucleus? Our work with the invertebrate animal model Caenorhabditis elegans has demonstrated that this roundworm is exceptional because it does not synthesize heme but rather utilizes environmental heme to manufacture heme-containing proteins that have human homologs. Genomic screens in C. elegans identified several novel genes which we termed Heme Responsive Genes (HRGs). HRG-1, the first metazoan heme importer/transporter, is a permease with four transmembrane domains, and is conserved in vertebrates. Depletion of hrg-1 paralogs in worms causes disruption of organismal heme sensing. Zebrafish with lower levels of hrg1 reveals hydrocephalus, yolk tube malformations, and profound defects in erythropoiesis. Hrg1 is strongly expressed in macrophages of the reticuloendothelial system in human and mouse tissues and is upregulated in a time- and dose-dependent manner by either erythrophagocytosis (EP) or induction of hemolytic anemia in mice. Hrg1 traffics to the phagolysosomal membranes during EP and depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment, concomitant with suppression of heme degradation and iron export. Importantly, studies using yeast, zebrafish, and mouse macrophages reveal that specific missense polymorphisms in Hrg1 are defective in heme transport. To uncover additional molecules in organismal heme homeostasis, we conducted a genome-wide RNAi screen in live, transgenic heme-sensor worms. The screen identified several hundred new candidates that integrate signals from different tissues to regulate systemic heme homeostasis. Our findings represent major discoveries in heme trafficking and establish a paradigm for heme transport in animals. Disclosures:Hamza:Rakta Therapeutics: CEO Other, Consultancy.

Octopus arm regeneration: Role of acetylcholinesterase during morphological modification

The ability to regenerate whole-body structures has been long studied in both vertebrate and invertebrate animal models. Due to this regeneration capability here we propose the use of the Cephalopod Octopus vulgaris as a model of regeneration. We investigated the involvement of acetylcholinesterase (AChE) in the octopus arm regeneration. AChE has been demonstrated to have non-cholinergic functions in various cell types and to be involved in the regulation of cell proliferation, differentiation and apoptosis. In order to follow cell replacement in the octopus arm, we first assessed the expression of specific markers involved in cellular proliferation (AgNOR and PCNA). We showed that the activity of the enzyme AChE is related to the proliferation stage of the arm regenerative process. In the very initial stages of regrowth when most of the proliferation activity was at the level of the ‘blastema’ the cholinesterase activity was very low. AChE activity climbed slowly during the subsequent phase of cellular multiplication and, by the onset of morphogenesis, the activity rose sharply and active myogenesis was observed. AChE activity decreased then till reaching basal level at the time when the process of histogenesis occurred and the reestablishment of all the structures became evident. Interestingly AgNOR and AChE assay showed a similar trend in particular during the stages when the morphogenesis was mostly dependent upon cell proliferation. We suggest that AChE protein may have an important influence in the process of regeneration and that it could be considered as a potential target to promote or regulate the regenerative process.

Gene expression analyses of a regenerative cardiac model system in Ciona intestinalis

Ciona intestinalis is a useful invertebrate animal model system for studying the role of evolutionarily conserved genes among Chordates. In contrast to most vertebrates, Ciona hearts have the capacity for regeneration throughout their lifespan. An injury model system was used to stimulate regeneration in Ciona hearts. In order to identify genes that are involved in regeneration of the Ciona heart, microarray analysis was conducted on RNA from adult hearts with normal or damaged myocardium using custom Affymetrix GeneChips. Initial results indicate significant changes in the expression of 223 genes in hearts damaged by ligation in comparison to control hearts (fold change >;2, p<0.01, Student's t‐test) with limited false discovery (5.8%). Among these 223 genes, 117 have known orthologs to vertebrates, of which 68 genes were up‐regulated and 49 genes were down‐regulated. Altered orthologous genes were validated by RT‐PCR. In addition, expression patterns of genes identified in the microarray study are currently being identified via in situ hybridization in juvenile and adult Ciona hearts. Genes of interest include: ciFGF 9/16/20, ciFoxO, and ciFoxP. In combination, these studies will help to elucidate the genetic basis of myocardial regeneration in Ciona and provide further insight into the role of orthologous genes in the regulation of cardiac myocyte proliferation.Grant Funding Source: NIH R15 Area Grant

Peroxisome deficient invertebrate and vertebrate animal models.

Although peroxisomes are ubiquitous organelles in all animal species, their importance for the functioning of tissues and organs remains largely unresolved. Because peroxins are essential for the biogenesis of peroxisomes, an obvious approach to investigate their physiological role is to inactivate a Pex gene or to suppress its translation. This has been performed in mice but also in more primitive organisms including D. melanogaster, C. elegans, and D. rerio, and the major findings and abnormalities in these models will be highlighted. Although peroxisomes are generally not essential for embryonic development and organogenesis, a generalized inactivity of peroxisomes affects lifespan and posthatching/postnatal growth, proving that peroxisomal metabolism is necessary for the normal maturation of these organisms. Strikingly, despite the wide variety of model organisms, corresponding tissues are affected including the central nervous system and the testis. By inactivating peroxisomes in a cell type selective way in the brain of mice, it was also demonstrated that peroxisomes are necessary to prevent neurodegeneration. As these peroxisome deficient model organisms recapitulate pathologies of patients affected with peroxisomal diseases, their further analysis will contribute to the elucidation of still elusive pathogenic mechanisms.

Invertebrate models of dystonia.

The neurological movement disorder dystonia is an umbrella term for a heterogeneous group of related conditions where at least 20 monogenic forms have been identified. Despite the substantial advances resulting from the identification of these loci, the function of many DYT gene products remains unclear. Comparative genomics using simple animal models to examine the evolutionarily conserved functional relationships with monogenic dystonias represents a rapid route toward a comprehensive understanding of these movement disorders. Current studies using the invertebrate animal models Caenorhabditis elegans and Drosophila melanogaster are uncovering cellular functions and mechanisms associated with mutant forms of the well-conserved gene products corresponding to DYT1, DYT5a, DYT5b, and DYT12 dystonias. Here we review recent findings from the invertebrate literature pertaining to molecular mechanisms of these gene products, torsinA, GTP cyclohydrolase I, tyrosine hydroxylase, and the alpha subunit of Na+/K ATPase, respectively. In each study, the application of powerful genetic tools developed over decades of intensive work with both of these invertebrate systems has led to mechanistic insights into these human disorders. These models are particularly amenable to large-scale genetic screens for modifiers or additional alleles, which are bolstering our understanding of the molecular functions associated with these gene products. Moreover, the use of invertebrate models for the evaluation of DYT genetic loci and their genetic interaction networks has predictive value and can provide a path forward for therapeutic intervention.

Urotensin II in Invertebrates: From Structure to Function in Aplysia californica

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

Molecular responses to stress conditions in invertebrate and vertebrate animal models

Molecular responses to stress conditions in invertebrate and vertebrate animal models

The stationary phase sigma factor, RpoS, regulates the production of a carbapenem antibiotic, a bioactive prodigiosin and virulence in the enterobacterial pathogen Serratia sp. ATCC 39006

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and invertebrate animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes pectate lyase and cellulase. We showed previously that deletion of the RNA chaperone Hfq abolished antibiotic production and attenuated virulence in both animal and plant hosts. Hfq and dependent small RNAs (sRNAs) are known to regulate the post-transcriptional expression of rpoS, which encodes σ(S), the stationary phase sigma factor subunit of RNA polymerase. An S39006 hfq deletion mutant showed decreased transcript levels of rpoS. Therefore, in this study we investigated whether the phenotypes regulated by Hfq were mediated through its control of rpoS. Whereas loss of Hfq abolished prodigiosin and carbapenem production and attenuated virulence in both C. elegans and potato, characterization of an S39006 rpoS mutant showed unexpectedly elevated prodigiosin and carbapenem production. Furthermore, the rpoS mutant exhibited attenuated animal pathogenesis, but not plant pathogenesis. Additionally, a homologue of the Hfq-dependent sRNA, RprA, was identified and shown to regulate prodigiosin production in a manner consistent with its role in positively regulating translation of rpoS mRNA. Combined, these results demonstrate that Hfq regulation of secondary metabolism and plant pathogenesis is independent of RpoS and establishes RpoS and RprA as regulators of antibiotic production.

Animal welfare and use of silkworm as a model animal

Sacrificing model animals is required for developing effective drugs before being used in human beings. In Japan today, at least 4,210,000 mice and other mammals are sacrificed to a total of 6,140,000 per year for the purpose of medical studies. All the animals treated in Japan, including test animals, are managed under control of "Act on Welfare and Management of Animals". Under the principle of this Act, no person shall kill, injure, or inflict cruelty on animals without due cause. "Animal" addressed in the Act can be defined as a "vertebrate animal". If we can make use of invertebrate animals in testing instead of vertebrate ones, that would be a remarkable solution for the issue of animal welfare. Furthermore, there are numerous advantages of using invertebrate animal models: less space and small equipment are enough for taking care of a large number of animals and thus are cost-effective, they can be easily handled, and many biological processes and genes are conserved between mammals and invertebrates. Today, many invertebrates have been used as animal models, but silkworms have many beneficial traits compared to mammals as well as other insects. In a Genome Pharmaceutical Institute's study, we were able to achieve a lot making use of silkworms as model animals. We would like to suggest that pharmaceutical companies and institutes consider the use of the silkworm as a model animal which is efficacious both for financial value by cost cutting and ethical aspects in animals' welfare.

Tasco(®), a product of Ascophyllum nodosum, imparts thermal stress tolerance in Caenorhabditis elegans.

Tasco®, a commercial product manufactured from the brown alga Ascophyllum nodosum, has been shown to impart thermal stress tolerance in animals. We investigated the physiological, biochemical and molecular bases of this induced thermal stress tolerance using the invertebrate animal model, Caenorhabiditis elegans. Tasco® water extract (TWE) at 300 μg/mL significantly enhanced thermal stress tolerance as well as extended the life span of C. elegans. The mean survival rate of the model animals under thermal stress (35 °C) treated with 300 μg/mL and 600 μg/mL TWE, respectively, was 68% and 71% higher than the control animals. However, the TWE treatments did not affect the nematode body length, fertility or the cellular localization of daf-16. On the contrary, TWE under thermal stress significantly increased the pharyngeal pumping rate in treated animals compared to the control. Treatment with TWE also showed differential protein expression profiles over control following 2D gel-electrophoresis analysis. Furthermore, TWE significantly altered the expression of at least 40 proteins under thermal stress; among these proteins 34 were up-regulated while six were down-regulated. Mass spectroscopy analysis of the proteins altered by TWE treatment revealed that these proteins were related to heat stress tolerance, energy metabolism and a muscle structure related protein. Among them heat shock proteins, superoxide dismutase, glutathione peroxidase, aldehyde dehydrogenase, saposin-like proteins 20, myosin regulatory light chain 1, cytochrome c oxidase RAS-like, GTP-binding protein RHO A, OS were significantly up-regulated, while eukaryotic translation initiation factor 5A-1 OS, 60S ribosomal protein L18 OS, peroxiredoxin protein 2 were down regulated by TWE treatment. These results were further validated by gene expression and reporter gene expression analyses. Overall results indicate that the water soluble components of Tasco® imparted thermal stress tolerance in the C. elegans by altering stress related biochemical pathways.

Post-transcriptional gene silencing by RNA interference in non-mammalian vertebrate systems: Where do we stand?

RNA interference (RNAi), the process by which double stranded RNA induces the silencing of endogenous genes through the degradation of its correspondent messenger RNA, has been used for post-transcriptional gene silencing allowing scientists to better understand gene function, becoming a powerful tool in reverse genetics for in vivo and in vitro systems. Successful results in vivo have been obtained from invertebrate animal models, whereas vertebrate systems have been limited primarily to mammalian models and cell lines. Nevertheless, exciting results have also been reported from non-mammalian vertebrate models, such as the knock-down of endogenous genes in Xenopus tadpoles by a construct containing both a Xenopus-specific shRNA sequence and the human Ago2 (which is a key enzyme in the RNAi silencing complex), or the design of a novel vector expressing a miRNA driven by a tissue-specific promoter in zebrafish, and the use of an avian retroviral vector to deliver miRNA and shRNA in chicken embryos proving to be effective in knocking-down endogenous genes with a long lasting effect, to mention some examples. Whether dsRNA is able to initiate a specific RNAi response, or all the factors required for RNAi are present in non-mammalian vertebrates, are still questions which remain to be answered. Further progress in understanding natural RNAi mechanisms in non-mammalian vertebrates will help scientists to overcome difficulties and improve this gene silencing technology. There is no doubt that in few years RNAi silencing approaches will become the tool of choice to knock-down genes in all groups of non-mammalian vertebrates, fulfilling different purposes, from basic research to animal therapeutics and drug discovery.

Invertebrate Animal Models as Tools to Study Fungal Pathogenesis and Treatment

Invertebrate Animal Models as Tools to Study Fungal Pathogenesis and Treatment