Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome.

BACKGROUND/OBJECTIVES: Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. RESULTS: The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. CONCLUSION: In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

Intrasexual cuticular hydrocarbon dimorphism in a wasp sheds light on hydrocarbon biosynthesis genes in Hymenoptera.

Cuticular hydrocarbons (CHCs) cover the cuticle of insects and serve as desiccation barrier and as semiochemicals. While the main enzymatic steps of CHC biosynthesis are well understood, few of the underlying genes have been identified. Here we show how exploitation of intrasexual CHC dimorphism in a mason wasp, Odynerus spinipes, in combination with whole-genome sequencing and comparative transcriptomics facilitated identification of such genes. RNAi-mediated knockdown of twelve candidate gene orthologs in the honey bee, Apis mellifera, confirmed nine genes impacting CHC profile composition. Most of them have predicted functions consistent with current knowledge of CHC metabolism. However, we found first-time evidence for a fatty acid amide hydrolase also influencing CHC profile composition. In situ hybridization experiments furthermore suggest trophocytes participating in CHC biosynthesis. Our results set the base for experimental CHC profile manipulation in Hymenoptera and imply that the evolutionary origin of CHC biosynthesis predates the arthropods' colonization of land.

Microarray expression profiling of fndc3a zebrafish mutants.

The group of Fibronectin type III domain-containing (FNDC; InterPro IPR003961) protein super family splits into a large number of gene-orthologues and mediates a variety of cellular functions during development and disease. They act as anti-inflammatory factors, are linked to cell-cell-interactions, regulate cell signaling and are associated with different cancer types, like cervical and colorectal. One member of this gene family is FNDC3A , which influences different developmental processes in vertebrates, like Sertoli cell/spermatid adhesion in mice testis, bone traits in chicken, and fin development in zebrafish. To identify downstream molecular processes during vertebrate development we investigated gene expression profiles in the previously established fndc3a zebrafish mutants via microarray analyses on 22 hpf embryos (26-somite stage). Our analyses imply distinct transcriptional profiles between genotype groups and hint to altered cell binding and catalytic activity in fndc3a mutants.

Altered Expression of TMEM43 Causes Abnormal Cardiac Structure and Function in Zebrafish.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.

Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing.

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Potentially inappropriate medications and medication combinations before, during and after hospitalizations: an analysis of pathways and determinants in the Swiss healthcare setting.

BACKGROUND: A hospitalization phase represents a challenge to medication safety especially for multimorbid patients as acute medical needs might interact with pre-existing medications or evoke adverse drug effects. This project aimed to examine the prevalence and risk factors of potentially inappropriate medications (PIMs) and medication combinations (PIMCs) in the context of hospitalizations. METHODS: Analyses are based on claims data of patients (≥65 years) with basic mandatory health insurance at the Helsana Group, and on data from the Hirslanden Swiss Hospital Group. We assessed PIMs and PIMCs of patients who were hospitalized in 2013 at three different time points (quarter prior, during, after hospitalization). PIMs were identified using the PRISCUS list, whereas PIMCs were derived from compendium.ch. Zero-inflated Poisson regression models were applied to determine risk factors of PIMs and PIMCs. RESULTS: Throughout the observation period, more than 80% of patients had at least one PIM, ranging from 49.7% in the pre-hospitalization, 53.6% in the hospitalization to 48.2% in the post-hospitalization period. PIMCs were found in 46.6% of patients prior to hospitalization, in 21.3% during hospitalization, and in 25.0% of patients after discharge. Additional medication prescriptions compared to the preceding period and increasing age were the main risk factors, whereas managed care was associated with a decrease in PIMs and PIMCs. CONCLUSION: We conclude that a patient's hospitalization offers the possibility to increase medication safety. Nevertheless, the prevalence of PIMs and PIMCs is relatively high in the study population. Therefore, our results indicate a need for interventions to increase medication safety in the Swiss healthcare setting.

Exploration of zebrafish larvae as an alternative whole-animal model for nephrotoxicity testing.

Due to an increasing demand for testing of new and existing chemicals and legal restrictions for the use of animals, there is a strong need for alternative approaches to assess systemic toxicity. Embryonic and larval zebrafish (Danio rerio) are increasingly recognized as a promising alternative whole-animal model that may be able to overcome limitations of cell-based in vitro assays and bridge the gap between high-throughput in vitro screening and low-throughput in vivo tests in animals. Despite the relatively simple anatomical structure of the zebrafish larval kidney (pronephros) - composed of only two nephrons - the pronephros shares major functions and cell types with mammalian nephrons. Glomerular filtration begins at 48 h post fertilization. The aim of the present study was to investigate if early zebrafish larvae might be a suitable model for nephrotoxicity testing. On day 3 post fertilization, larval zebrafish were treated with selected nephrotoxins (aristolochic acid, cadmium chloride, potassium bromate, ochratoxin A, gentamicin) for 48 h. Histological evaluation of zebrafish larvae exposed to model nephrotoxins revealed tubule injury as evidenced by dilated tubules with loss of the brush border, tubule cell necrosis and disorganization of the tubular epithelium. These changes were most severe after treatment with gentamicin, which also impaired pronephros function as evidenced by reduced clearance of FITC-dextran. Whole-mount in situ hybridization showing loss of cdh17 expression revealed site-specific injury to the proximal tubule segment. Analysis of genes previously identified as novel biomarkers of kidney injury in mammals showed upregulation of the kidney injury marker genes heme oxygenase 1 (hmox1), clusterin (clu), secreted phosphoprotein/osteopontin (spp1), connective tissue growth factor (ctgf) and kim-1 (havcr-1) in response to nephrotoxin treatment, although the response of individual genes varied across compounds. Consistent with the severity of lesions and impaired kidney function, the most prominent gene expression changes occurred in larvae exposed to gentamicin. Overall, our results suggest that larval zebrafish may be a suitable alternative model organism for nephrotoxicity screening, yet further improvements and integration with quantitative in vitro to in vivo extrapolation will be needed to predict human toxicity.

TNAP as a New Player in Chronic Inflammatory Conditions and Metabolism.

This review summarizes important information on the ectoenzyme tissue-nonspecific alkaline phosphatase (TNAP) and gives a brief insight into the symptoms, diagnostics, and treatment of the rare disease Hypophosphatasia (HPP), which is resulting from mutations in the TNAP encoding ALPL gene. We emphasize the role of TNAP beyond its well-known contribution to mineralization processes. Therefore, above all, the impact of the enzyme on central molecular processes in the nervous system and on inflammation is presented here.

Tissue-Nonspecific Alkaline Phosphatase-A Gatekeeper of Physiological Conditions in Health and a Modulator of Biological Environments in Disease.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the ALPL gene and consequently altered TNAP activity are suffering from the rare metabolic disease hypophosphatasia (HPP). This systemic disease is mainly characterized by impaired bone and dental mineralization but may also be accompanied by neurological symptoms, like anxiety disorders, seizures, and depression. HPP characteristically affects all ages and shows a wide range of clinical symptoms and disease severity, which results in the classification into different clinical subtypes. This review describes the molecular function of TNAP during the mineralization of bones and teeth, further discusses the current knowledge on the enzyme's role in the nervous system and in sensory perception. An additional focus is set on the molecular role of TNAP in health and on functional observations reported in common laboratory vertebrate disease models, like rodents and zebrafish.

Investigation of alpl expression and Tnap-activity in zebrafish implies conserved functions during skeletal and neuronal development.

Hypophosphatasia (HPP) is a rare genetic disease with diverse symptoms and a heterogeneous severity of onset with underlying mutations in the ALPL gene encoding the ectoenzyme Tissue-nonspecific alkaline phosphatase (TNAP). Considering the establishment of zebrafish (Danio rerio) as a new model organism for HPP, the aim of the study was the spatial and temporal analysis of alpl expression in embryos and adult brains. Additionally, we determined functional consequences of Tnap inhibition on neural and skeletal development in zebrafish. We show that expression of alpl is present during embryonic stages and in adult neuronal tissues. Analyses of enzyme function reveal zones of pronounced Tnap-activity within the telencephalon and the mesencephalon. Treatment of zebrafish embryos with chemical Tnap inhibitors followed by axonal and cartilage/mineralized tissue staining imply functional consequences of Tnap deficiency on neuronal and skeletal development. Based on the results from neuronal and skeletal tissue analyses, which demonstrate an evolutionary conserved role of this enzyme, we consider zebrafish as a promising species for modeling HPP in order to discover new potential therapy strategies in the long-term.

Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion.

The transport and Golgi organization 1 (TANGO1) proteins play pivotal roles in the secretory pathway. Full length TANGO1 is a transmembrane protein localised at endoplasmic reticulum (ER) exit sites, where it binds bulky cargo within the ER lumen and recruits membranes from the ER Golgi intermediate compartment to create an exit route for their export. Here we report the first TANGO1-associated syndrome in humans. A synonymous substitution that results in exon eight skipping in most mRNA molecules, ultimately leading to a truncated TANGO1 protein was identified as disease-causing mutation. The four homozygously affected sons of a consanguineous family display severe dentinogenesis imperfecta, short stature, various skeletal abnormalities, insulin-dependent diabetes mellitus, sensorineural hearing loss, and mild intellectual disability. Functional studies in HeLa and U2OS cells revealed that the corresponding truncated TANGO1 protein is dispersed in the ER and its expression in cells with intact endogenous TANGO1 impairs cellular collagen I secretion.

Novel Loss-of-Function Variants in CDC14A are Associated with Recessive Sensorineural Hearing Loss in Iranian and Pakistani Patients.

CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.

ECM alterations in Fndc3a (Fibronectin Domain Containing Protein 3A) deficient zebrafish cause temporal fin development and regeneration defects.

Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3awue1/wue1) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3awue1/wue1 mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish.

On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio).

The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish.

Mutations in ILK, encoding integrin-linked kinase, are associated with arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy is a genetic heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes leading to life-threatening ventricular arrhythmias, heart failure, and sudden cardiac death. Mutations in genes encoding cardiac junctional proteins are known to cause about half of cases, while remaining genetic causes are unknown. Using exome sequencing, we identified 2 missense variants (p.H33N and p.H77Y) that were predicted to be damaging in the integrin-linked kinase (ILK) gene in 2 unrelated families. The p.H33N variant was found to be de novo. ILK links integrins and the actin cytoskeleton, and is essential for the maintenance of normal cardiac function. Both of the new variants are located in the ILK ankyrin repeat domain, which binds to the first LIM domain of the adaptor proteins PINCH1 and PINCH2. In silico binding studies proposed that the human variants disrupt the ILK-PINCH complex. Recombinant mutant ILK expressed in H9c2 rat myoblast cells shows aberrant prominent cytoplasmic localization compared to the wild-type. Expression of human wild-type and mutant ILK under the control of the cardiac-specific cmlc2 promotor in zebrafish shows that p.H77Y and p.P70L, a variant previously reported in a dilated cardiomyopathy family, cause cardiac dysfunction and death by about 2-3 weeks of age. Our findings provide genetic and functional evidence that ILK is a cardiomyopathy disease gene and highlight its relevance for diagnosis and genetic counseling of inherited cardiomyopathies.

Xmrk-induced melanoma progression is affected by Sdf1 signals through Cxcr7.

Chemokine signals mediated by Sdf1/Cxcl12 through the chemokine receptor Cxcr4 are thought to play an instructive role in tumor migration and organ-specific metastasis. We have used a small aquarium fish model to contribute to a better understanding of how the course of melanoma development is influenced by Sdf1 signals in vivo. We studied oncogene-induced skin tumor appearance and progression in the transgenic medaka (Oryzias latipes) melanoma model. Similar to humans, invasive medaka melanomas show increased levels of sdf1, cxcr4, and cxcr7 gene expression. Stable transgenic fish lines overexpressing sdf1 exclusively in pigment cells showed a reduction in melanoma appearance and progression. Remarkably, diminished levels of functional Cxcr7, but not of Cxcr4b, resulted in strongly reduced melanoma invasiveness and a repression of melanoma. Our results thereby indicate that Sdf1 signals via Cxcr7 are able to constrain melanoma growth in vivo and that these signals influence tumor outcome.

Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model.

Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifen-inducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.

snail gene expression in the medaka, Oryzias latipes.

Snail transcription factors have prominent roles during embryonic development of vertebrates. They are often involved in cell migration processes during neural crest development, epithelial-mesenchymal transition and cancer progression. Comparative expression studies of snai gene family members in different vertebrate species are expected to contribute to a better understanding of their roles during development and reflect their evolutionary history. To investigate and to compare the expression patterns of snai genes in a second main fish model we used the medaka fish (Oryzias latipes), a complementary teleost model to zebrafish. We identified three snai gene family members, snai1a, snai1b and snai2. Phylogenetic and synteny analysis show a close relatedness of all family members to other vertebrate snai genes. Surprisingly, no homologue of snai3 could be identified in medaka, although this gene is present in zebrafish and the puffer fishes. Here we demonstrate that while most expression domains of medaka snai genes are comparable to zebrafish, the contribution of the individual paralogs to the overall pattern differs between the two teleosts and indicate lineage specific expression shuffling.

Pigmentary function and evolution of tyrp1 gene duplicates in fish.

The function of the tyrosinase-related protein 1 (Tyrp1) has not yet been investigated in vertebrates basal to tetrapods. Teleost fishes have two duplicates of the tyrp1 gene. Here, we show that the teleost tyrp1 duplicates have distributed the ancestral gene expression in the retinal pigment epithelium (RPE) and melanophores in a species-specific manner. In medaka embryos, tyrp1a expression is found in the RPE and in melanophores while tyrp1b is only expressed in melanophores. In zebrafish embryos, expression of tyrp1 paralogs overlaps in the RPE and in melanophores. Knockdown of each zebrafish tyrp1 duplicate alone does not show pigmentary defects, but simultaneous knockdown of both tyrp1 genes results in the formation of brown instead of black eumelanin accompanied by severe melanosome defects. Our study suggests that the brown melanosome color in Tyrp1-deficient vertebrates is an effect of altered eumelanin synthesis. Black eumelanin formation essentially relies on the presence of Tyrp1 and some of its function is most likely conserved from the common ancestor of bony vertebrates.