OTX2 duplications: a recurrent cause of oculo-auriculo-vertebral spectrum.

BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is the second most common cause of head and neck malformations in children after orofacial clefts. OAVS is clinically heterogeneous and characterised by a broad range of clinical features including ear anomalies with or without hearing loss, hemifacial microsomia, orofacial clefts, ocular defects and vertebral abnormalities. Various genetic causes were associated with OAVS and copy number variations represent a recurrent cause of OAVS, but the responsible gene often remains elusive. METHODS: We described an international cohort of 17 patients, including 10 probands and 7 affected relatives, presenting with OAVS and carrying a 14q22.3 microduplication detected using chromosomal microarray analysis. For each patient, clinical data were collected using a detailed questionnaire addressed to the referring clinicians. We subsequently studied the effects of OTX2 overexpression in a zebrafish model. RESULTS: We defined a 272 kb minimal common region that only overlaps with the OTX2 gene. Head and face defects with a predominance of ear malformations were present in 100% of patients. The variability in expressivity was significant, ranging from simple chondromas to severe microtia, even between intrafamilial cases. Heterologous overexpression of OTX2 in zebrafish embryos showed significant effects on early development with alterations in craniofacial development. CONCLUSIONS: Our results indicate that proper OTX2 dosage seems to be critical for the normal development of the first and second branchial arches. Overall, we demonstrated that OTX2 genomic duplications are a recurrent cause of OAVS marked by auricular malformations of variable severity.

The Dct-/- Mouse Model to Unravel Retinogenesis Misregulation in Patients with Albinism.

We have recently identified DCT encoding dopachrome tautomerase (DCT) as the eighth gene for oculocutaneous albinism (OCA). Patients with loss of function of DCT suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in Dct-/- mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages, contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanization. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of Dct-/- newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient Tyrc/c embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in Dct-/- postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The Dct-/- mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.

Oculo-auriculo-vertebral spectrum: new genes and literature review on a complex disease.

Oculo-auriculo-vertebral spectrum (OAVS) or Goldenhar syndrome is due to an abnormal development of first and second branchial arches derivatives during embryogenesis and is characterised by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical and genetic heterogeneity of this spectrum with incomplete penetrance and variable expressivity, render its molecular diagnosis difficult. Only a few recurrent CNVs and genes have been identified as causatives in this complex disorder so far. Prenatal environmental causal factors have also been hypothesised. However, most of the patients remain without aetiology. In this review, we aim at updating clinical diagnostic criteria and describing genetic and non-genetic aetiologies, animal models as well as novel diagnostic tools and surgical management, in order to help and improve clinical care and genetic counselling of these patients and their families.

A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum.

Goldenhar syndrome or oculo-auriculo-vertebral spectrum (OAVS) is a complex developmental disorder characterized by asymmetric ear anomalies, hemifacial microsomia, ocular and vertebral defects. We aimed at identifying and characterizing a new gene associated with OAVS. Two affected brothers with OAVS were analyzed by exome sequencing that revealed a missense variant (p.(Asn358Ser)) in the EYA3 gene. EYA3 screening was then performed in 122 OAVS patients that identified the same variant in one individual from an unrelated family. Segregation assessment in both families showed incomplete penetrance and variable expressivity. We investigated this variant in cellular models to determine its pathogenicity and demonstrated an increased half-life of the mutated protein without impact on its ability to dephosphorylate H2AFX following DNA repair pathway induction. Proteomics performed on this cellular model revealed four significantly predicted upstream regulators which are PPARGC1B, YAP1, NFE2L2 and MYC. Moreover, eya3 knocked-down zebrafish embryos developed specific craniofacial abnormalities corroborating previous animal models and supporting its involvement in the OAVS. Additionally, EYA3 gene expression was deregulated in vitro by retinoic acid exposure. EYA3 is the second recurrent gene identified to be associated with OAVS. Moreover, based on protein interactions and related diseases, we suggest the DNA repair as a key molecular pathway involved in craniofacial development.

Dopachrome tautomerase variants in patients with oculocutaneous albinism.

PURPOSE: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. METHODS: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. RESULTS: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. CONCLUSION: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8.

Novel variants in the BLOC1S3 gene in patients presenting a mild form of Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) associates oculocutaneous albinism and systemic affections including platelet dense granules anomalies leading to bleeding diathesis and, depending on the form, pulmonary fibrosis, immunodeficiency, and/or granulomatous colitis. So far, 11 forms of autosomal recessive HPS caused by pathogenic variants in 11 different genes have been reported. We describe three HPS-8 consanguineous families with different homozygous pathogenic variants in BLOC1S3 (NM_212550.3), one of which is novel. These comprise two deletions leading to a reading frameshift (c.385_403del, c.338_341del) and one in frame deletion (c.444_467del). All patients have moderate oculocutaneous albinism and bleeding diathesis, but other HPS symptoms are not described. One patient diagnosed with HPS-8 suffered from lymphocyte-predominant Hodgkin lymphoma. The mild severity of HPS-8 is consistent with other HPS forms caused by variants in BLOC-1 complex coding genes (HPS-7, DTNBP1; HPS-9, BLOC1S6, HPS-11, BLOC1S5).

Functional and genetic analyses of ZYG11B provide evidences for its involvement in OAVS.

BACKGROUND: The Oculo-Auriculo-Vertebral Spectrum (OAVS) or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical heterogeneity of this spectrum and its incomplete penetrance limited the molecular diagnosis. In this study, we describe a novel causative gene, ZYG11B. METHODS: A sporadic case of OAVS was analyzed by whole exome sequencing in trio strategy. The identified candidate gene, ZYG11B, was screened in 143 patients by next generation sequencing. Overexpression and immunofluorescence of wild-type and mutated ZYG11B forms were performed in Hela cells. Moreover, morpholinos were used for transient knockdown of its homologue in zebrafish embryo. RESULTS: A nonsense de novo heterozygous variant in ZYG11B, (NM_024646, c.1609G>T, p.Glu537*) was identified in a single OAVS patient. This variant leads in vitro to a truncated protein whose subcellular localization is altered. Transient knockdown of the zebrafish homologue gene confirmed its role in craniofacial cartilages architecture and in notochord development. Moreover, ZYG11B expression regulates a cartilage master regulator, SOX6, and is regulated by Retinoic Acid, a known developmental toxic molecule leading to clinical features of OAVS. CONCLUSION: Based on genetic, cellular and animal model data, we proposed ZYG11B as a novel rare causative gene for OAVS.

Description of a family with X-linked oculo-auriculo-vertebral spectrum associated with polyalanine tract expansion in ZIC3.

Oculo-auriculo-vertebral spectrum (OAVS) [MIM:164210], or Goldenhar syndrome, is a developmental disorder associating defects of structures derived from the first and second branchial arches. The genetic origin of OAVS is supported by the description of rare deleterious variants in a few causative genes, and several chromosomal copy number variations. We describe here a large family with eight male members affected by a mild form of the spectrum, mostly auricular defects, harboring a hemizygous ZIC3 variant detected by familial exome sequencing: c.159_161dup p.(Ala55dup), resulting in an expansion of the normal 10 consecutive alanine residues to 11 alanines. Segregation analysis shows its presence in all the affected individuals, with a recessive X-linked transmission. Whole-genome sequencing performed in another affected male allowed to exclude linkage disequilibrium between this ZIC3 variant and another potential pathogenic variant in this family. Furthermore, by screening of a cohort of 274 OAVS patients, we found 1 male patient carrying an expansion of 10 to 12 alanines, a variant previously reported in patient presenting with VACTERL. Loss-of-function variants of ZIC3 are causing heterotaxy or cardiac malformations. These alanine expansion variants could have a different impact on the protein and thereby resulting in a different phenotype within the OAVS/VACTERL.

BLOC1S5 pathogenic variants cause a new type of Hermansky-Pudlak syndrome.

PURPOSE: Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, excessive bleeding, and often additional symptoms. Variants in ten different genes have been involved in HPS. However, some patients lack variants in these genes. We aimed to identify new genes involved in nonsyndromic or syndromic forms of albinism. METHODS: Two hundred thirty albinism patients lacking a molecular diagnosis of albinism were screened for pathogenic variants in candidate genes with known links to pigmentation or HPS pathophysiology. RESULTS: We identified two unrelated patients with distinct homozygous variants of the BLOC1S5 gene. Patients had mild oculocutaneous albinism, moderate bleeding diathesis, platelet aggregation deficit, and a dramatically decreased number of platelet dense granules, all signs compatible with HPS. Functional tests performed on platelets of one patient displayed an absence of the obligate multisubunit complex BLOC-1, showing that the variant disrupts BLOC1S5 function and impairs BLOC-1 assembly. Expression of the patient-derived BLOC1S5 deletion in nonpigmented murine Bloc1s5-/- melan-mu melanocytes failed to rescue pigmentation, the assembly of a functional BLOC-1 complex, and melanosome cargo trafficking, unlike the wild-type allele. CONCLUSION: Mutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of Hermansky-Pudlak syndrome, HPS-11.

Deletion in 2q35 excluding the IHH gene leads to fetal severe limb anomalies and suggests a disruption of chromatin architecture.

The organization of mammalian genomes into sub-megabase sized Topologically Associated Domains (TADs) has recently been revealed by techniques derived from Chromosome Conformation Capture (3 C), such as High Chromosome Contact map (Hi-C). Disruption of this organization by structural variations can lead to ectopic interactions between enhancers and promoters, and to alteration of genes expression patterns. This mechanism has already been described as the main pathophysiological mechanism in several syndromes with congenital malformations. We describe here the case of a fetus with a severe multiple congenital anomalies syndrome, including extensive polydactyly of the four limbs. This fetus carries a de novo deletion next to the IHH gene, encompassing a TAD boundary. Such an IHH TAD boundary deletion has already been described in the Dbf mouse model, which shows a quite similar, but less severe phenotype. We hypothesize that the deletion harbored by this fetus results in the same pathophysiological mechanisms as those of the Dbf model. The description of this case expands the spectrum of the disruption of chromatin architecture of WNT6/IHH/EPHA4/PAX3 locus, and could help to understand the mechanisms of chromatin interactions at this locus.

A novel de novo mutation in MYT1, the unique OAVS gene identified so far.

Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder characterized by hemifacial microsomia associated with ear, eyes and vertebrae malformations showing highly variable expressivity. Recently, MYT1, encoding the myelin transcription factor 1, was reported as the first gene involved in OAVS, within the retinoic acid (RA) pathway. Fifty-seven OAVS patients originating from Brazil were screened for MYT1 variants. A novel de novo missense variant affecting function, c.323C>T (p.(Ser108Leu)), was identified in MYT1, in a patient presenting with a severe form of OAVS. Functional studies showed that MYT1 overexpression downregulated all RA receptors genes (RARA, RARB, RARG), involved in RA-mediated transcription, whereas no effect was observed on CYP26A1 expression, the major enzyme involved in RA degradation, Moreover, MYT1 variants impacted significantly the expression of these genes, further supporting their pathogenicity. In conclusion, a third variant affecting function in MYT1 was identified as a cause of OAVS. Furthermore, we confirmed MYT1 connection to RA signaling pathway.

Functional validation of ABHD12 mutations in the neurodegenerative disease PHARC.

ABHD12 mutations have been linked to neurodegenerative PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract), a rare, progressive, autosomal, recessive disease. Although ABHD12 is suspected to play a role in the lysophosphatidylserine and/or endocannabinoid pathways, its precise functional role(s) leading to PHARC disease had not previously been characterized. Cell and zebrafish models were designed to demonstrate the causal link between an identified new missense mutation p.T253R, characterized in ABHD12 from a young patient, the previously characterized p.T202I and p.R352* mutations, and the associated PHARC. Measuring ABHD12 monoacylglycerol lipase activity in transfected HEK293 cells demonstrated inhibition with mutated isoforms. Both the expression pattern of zebrafish abhd12 and the phenotype of specific antisense morpholino oligonucleotide gene knockdown morphants were consistent with human PHARC hallmarks. High abhd12 transcript levels were found in the optic tectum and tract, colocalized with myelin basic protein, and in the spinal cord. Morphants have myelination defects and concomitant functional deficits, characterized by progressive ataxia and motor skill impairment. A disruption of retina architecture and retinotectal projections was observed, together with an inhibition of lens clarification and a low number of mechanosensory hair cells in the inner ear and lateral line system. The severe phenotypes in abhd12 knockdown morphants were rescued by introducing wild-type human ABHD12 mRNA, but not by mutation-harboring mRNAs. Zebrafish may provide a suitable vertebrate model for ABHD12 insufficiency and the study of functional impairment and potential therapeutic rescue of this rare, neurodegenerative disease.

Mutations in MYT1, encoding the myelin transcription factor 1, are a rare cause of OAVS.

BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder involving first and second branchial arches derivatives, mainly characterised by asymmetric ear anomalies, hemifacial microsomia, ocular defects and vertebral malformations. Although numerous chromosomal abnormalities have been associated with OAVS, no causative gene has been identified so far. OBJECTIVES: We aimed to identify the first causative gene for OAVS. METHODS: As sporadic cases are mostly described in Goldenhar syndrome, we have performed whole exome sequencing (WES) on selected affected individuals and their unaffected parents, looking for de novo mutations. Candidate gene was tested through transient knockdown experiment in zebrafish using a morpholino-based approach. A functional test was developed in cell culture in order to assess deleterious consequences of mutations. RESULTS: By WES, we identified a heterozygous nonsense mutation in one patient in the myelin transcription factor 1 (MYT1) gene. Further, we detected one heterozygous missense mutation in another patient among a cohort of 169 patients with OAVS. This gene encodes the MYT1. Functional studies by transient knockdown of myt1a, homologue of MYT1 in zebrafish, led to specific craniofacial cartilage alterations. Treatment with all-trans retinoic acid (RA), a known teratogenic agent causing OAVS, led to an upregulation of cellular endogenous MYT1 expression. Additionally, cellular wild-type MYT1 overexpression induced a downregulation of RA receptor ß (RARB), whereas mutated MYT1 did not. CONCLUSION: We report MYT1 as the first gene implicated in OAVS, within the RA signalling pathway.

Endocrinology: advances through omics and related technologies.

The rapid development of new omics technologies to measure changes at genetic, transcriptomic, proteomic, and metabolomics levels together with the evolution of methods to analyze and integrate the data at a systems level are revolutionizing the study of biological processes. Here we discuss how new approaches using omics technologies have expanded our knowledge especially in nontraditional models. Our increasing knowledge of these interactions and evolutionary pathway conservation facilitates the use of nontraditional species, both invertebrate and vertebrate, as new model species for biological and endocrinology research. The increasing availability of technology to create organisms overexpressing key genes in endocrine function allows manipulation of complex regulatory networks such as growth hormone (GH) in transgenic fish where disregulation of GH production to produce larger fish has also permitted exploration of the role that GH plays in testis development, suggesting that it does so through interactions with insulin-like growth factors. The availability of omics tools to monitor changes at nearly any level in any organism, manipulate gene expression and behavior, and integrate data across biological levels, provides novel opportunities to explore endocrine function across many species and understand the complex roles that key genes play in different aspects of the endocrine function.

A rapid transcriptome response is associated with desiccation resistance in aerially-exposed killifish embryos.

Delayed hatching is a form of dormancy evolved in some amphibian and fish embryos to cope with environmental conditions transiently hostile to the survival of hatchlings or larvae. While diapause and cryptobiosis have been extensively studied in several animals, very little is known concerning the molecular mechanisms involved in the sensing and response of fish embryos to environmental cues. Embryos of the euryhaline killifish Fundulus heteroclitus advance dvelopment when exposed to air but hatching is suspended until flooding with seawater. Here, we investigated how transcriptome regulation underpins this adaptive response by examining changes in gene expression profiles of aerially incubated killifish embryos at ∼100% relative humidity, compared to embryos continuously flooded in water. The results confirm that mid-gastrula embryos are able to stimulate development in response to aerial incubation, which is accompanied by the differential expression of at least 806 distinct genes during a 24 h period. Most of these genes (∼70%) appear to be differentially expressed within 3 h of aerial exposure, suggesting a broad and rapid transcriptomic response. This response seems to include an early sensing phase, which overlaps with a tissue remodeling and activation of embryonic development phase involving many regulatory and metabolic pathways. Interestingly, we found fast (0.5-1 h) transcriptional differences in representatives of classical "stress" proteins, such as some molecular chaperones, members of signalling pathways typically involved in the transduction of sensor signals to stress response genes, and oxidative stress-related proteins, similar to that described in other animals undergoing dormancy, diapause or desiccation. To our knowledge, these data represent the first transcriptional profiling of molecular processes associated with desiccation resistance during delayed hatching in non-mammalian vertebrates. The exceptional transcriptomic plasticity observed in killifish embryos provides an important insight as to how the embryos are able to rapidly adapt to non-lethal desiccation conditions.

Primary oocyte transcriptional activation of aqp1ab by the nuclear progestin receptor determines the pelagic egg phenotype of marine teleosts.

In marine teleosts, the aqp1ab water channel plays a vital role in the development of the pelagic egg phenotype. However, the developmental control of aqp1ab activation during oogenesis remains to be established. Here, we report the isolation of the 5'-flanking region of the teleost gilthead seabream aqp1ab gene, in which we identify conserved cis-regulatory elements for the binding of the nuclear progestin receptor (Pgr) and members of the Sox family of transcription factors. Subcellular localization studies indicated that the Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oogonia, whereas in meiosis-arrested primary growth (pre-vitellogenic) oocytes, when aqp1ab mRNA and protein are first synthesized, the Pgr appears to be completely translocated from the ooplasm into the nucleus. By contrast, sox9b is highly expressed in more advanced oocytes, coinciding with a strong depletion of aqp1ab transcripts in the oocyte. Functional characterization of wild-type and mutated aqp1ab promoter constructs, using mammalian cells and Xenopus laevis oocytes, demonstrated that aqp1ab transcription is initiated by the Pgr, which is activated by the progestin 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), the natural ligand of the seabream Pgr. In vitro incubation of seabream primary ovarian explants with the follicle-stimulating hormone or 17,20ß-P confirmed that progestin-activated Pgr enhanced Aqp1ab synthesis via the aqp1ab promoter. However, transactivation assays in heterologous systems showed that Sox transcription factors can potentially modulate this mechanism. These data uncover the existence of an endocrine pathway involved in the early activation of a water channel necessary for egg formation in marine teleosts.

Vitellogenin expression in white adipose tissue in female teleost fish.

In most oviparous animal species, oocyte growth occurs via the uptake of plasma egg yolk precursors, predominantly vitellogenins (Vtg). These glycolipoproteins are members of the large lipid transfer protein superfamily and key players in reproduction. While the vertebrate liver has been demonstrated to synthesize large amounts of Vtg, mostly under 17beta-estradiol control, the ability of other tissues to express significant amounts of Vtg has not been conclusively demonstrated. RT-PCR revealed vtg1 transcripts in female zebrafish and rainbow trout white adipose tissue (WAT). It was also found to coexpress mtp, known to perform the intracellular lipidation of Vtg prior to secretion. The liver and pancreas markers apobb2 and ins, or ela2, respectively, were not expressed in adipocytes. Whole-mount in situ hybridization and in situ RT-PCR tests of histological sections revealed vtg1 signal in adipocytes, whereas no signal was detected in infiltrated pancreatic islets. Transcript expression of vtg1 was induced in WAT of 17beta-estradiol-treated males, and the transcript and corresponding protein were detected in the thin rim of cytoplasm surrounding the adipocyte. Real-time quantitative RT-PCR showed that rainbow trout perivisceral WAT vtg1 transcript levels were high during early compared to late vitellogenesis. Taking normalized mRNA levels and tissue somatic index into account, vtg1 transcript levels at the beginning of oocyte yolk deposition were approximately 45 times lower in WAT than in liver, and these levels were not correlated to plasma Vtg and 17beta-estradiol concentrations. These findings suggest that WAT Vtg is implicated in providing components to the ovary during the early stages of vitellogenesis.

Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis.

Thyroxine-immunofluorescence quantitative disruption test (TIQDT) was designed to provide a simple, rapid, alternative bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. This study demonstrated that zebrafish eleutheroembryos provided a suitable vertebrate model, not only for screening the potential thyroid disrupting effect of molecules, but also for estimating the potential hazards associated with exposure to chemicals directly impairing thyroxine (T4) synthesis. Amitrole, potassium perchlorate, potassium thiocyanate, methimazole (MMI), phloroglucinol, 6-propyl-2-thiouracil, ethylenethiourea, benzophenone-2, resorcinol, pyrazole, sulfamethoxazole, sodium bromide, mancozeb, and genistein were classified as thyroid gland function disruptors. Concordance between TIQDT on zebrafish and mammalian published data was very high and the physiological relevance of T4-intrafollicular content was clearly higher than regulation at the transcriptional level of tg or slc5a5. Moreover, concentration-response analysis provided information about the thyroid disrupting potency and hazard of selected positive compounds. Finally, the effect of perchlorate, but not MMI, was completely rescued by low-micromolar amounts of iodide. TIQDT performed on zebrafish eleutheroembryos is an alternative whole-organism screening assay that provides relevant information for environmental and human risk assessments.

Zebrafish obesogenic test: a tool for screening molecules that target adiposity.

Dietary and xenobiotic compounds may alter endocrine signaling and lipid homeostasis, thus inducing obesity. We describe a short-term assay method, the zebrafish obesogenic (ZO) test, for examining the effects of diet, drugs, and environmental contaminants, singly or in combination, on white adipose tissue (WAT) dynamics in live larvae. The ZO test is an intermediate step in obesity research, between in vitro and rodent assays, and may be also used to study the effect of environmental toxicants on the adiposity of aquatic species. The procedure, using Nile Red (NR) fluorescent probe to reveal adipocyte lipid droplets, is suitable for pharmaceutical or toxicological screening. Larvae treated at an environmentally-relevant concentration of tributyltin chloride (TBT), an environmental obesogen, exhibited a remarkable increase in adiposity, irrespective of the lipid composition of the background diet. Exogenous compounds, e.g., rosiglitazone or TBT, known to increase adiposity in the fasting state, were classified as obesogenic. Anti-obesogenic compounds favored a decrease in adiposity in the fasting state. The ZO test, using adipocyte lipid droplet size and adiposity as its endpoints, is a whole-organism alternative testing assay for obesogenic and anti-obesogenic compounds and mixtures and provides relevant information for environmental and human risk assessments.

Molecular and functional characterization of catfish (Heteropneustes fossilis) aquaporin-1b: changes in expression during ovarian development and hormone-induced follicular maturation.

The oocytes of the freshwater catfish Heteropneustes fossilis hydrate during hormone-induced meiotic maturation. To investigate if this process may be mediated by aquaporins (AQPs), as it occurs in marine fish producing highly hydrated eggs, the cloning of ovarian AQPs in catfish was carried out. Using degenerate primers for conserved domains of the major intrinsic protein (MIP) family, and 5' and 3'end amplification procedures, a full-length cDNA encoding for an AQP1-like protein was isolated. The predicted protein showed the typical six transmembrane domains and two Asn-Pro-Ala (NPA) motifs conserved among the members of the AQP superfamily. Phylogenetic analysis indicated that the catfish AQP clustered with the teleost-specific aquaporin-1b subfamily, and accordingly it was termed HfAqp1b. Heterologous expression in Xenopus laevis oocytes indicated that HfAqp1b encoded for a functional AQP, water permeability being enhanced by cAMP. Site-directed mutagenesis revealed that cAMP induced the translocation of HfAqp1b into the oocyte plasma membrane most likely through the phosphorylation of HfAqp1b Ser(227). In adult catfish, hfaqp1b transcripts were detected exclusively in ovary and brain and showed significant seasonal variations; in the ovary, hfaqp1b was maximally expressed during the pre-spawning period, whereas in the brain the highest expression was detected during spawning. In vitro stimulation of isolated catfish ovarian follicles with vasotocin (VT) or human chorionic gonadotropin (hCG), which induce oocyte maturation and hydration, elevated the hfaqp1b transcript levels after 6 or 16 h of incubation, respectively. These results suggest that HfAqp1b may play a role during VT- and hCG-induced oocyte hydration in catfish, and that VT may regulate HfAqp1b at the transcriptional and post-translational level in a manner similar to the vasopressin-dependent mammalian AQP2.