Genetic studies of intellectual disability and identification of monogenic causes of obesity in humans have made immense contribution toward the understanding of the brain and control of body mass. The leptin > melanocortin > SIM1 pathway is dysregulated in multiple monogenic human obesity syndromes but its downstream targets are still unknown. In ten individuals from six families, with overlapping 6q16.1 deletions, we describe a disorder of variable developmental delay, intellectual disability, and susceptibility to obesity and hyperphagia. The 6q16.1 deletions segregated with the phenotype in multiplex families and were shown to be de novo in four families, and there was dramatic phenotypic overlap among affected individuals who were independently ascertained without bias from clinical features. Analysis of the deletions revealed a ∼350 kb critical region on chromosome 6q16.1 that encompasses a gene for proneuronal transcription factor POU3F2, which is important for hypothalamic development and function. Using morpholino and mutant zebrafish models, we show that POU3F2 lies downstream of SIM1 and controls oxytocin expression in the hypothalamic neuroendocrine preoptic area. We show that this finding is consistent with the expression patterns of POU3F2 and related genes in the human brain. Our work helps to further delineate the neuro-endocrine control of energy balance/body mass and demonstrates that this molecular pathway is conserved across multiple species.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc) mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression.

CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.

Cell migration and invasion are essential processes for metastatic dissemination of cancer cells. Significant progress has been made in developing new therapies against oncogenic signaling to eliminate cancer cells and shrink tumors. However, inherent heterogeneity and treatment-induced adaptation to drugs commonly enable subsets of cancer cells to survive therapy. In addition to local recurrence, these cells escape a primary tumor and migrate through the stroma to access the circulation and metastasize to different organs, leading to an incurable disease. As such, therapeutics that block migration and invasion of cancer cells may inhibit or reduce metastasis and significantly improve cancer therapy. This is particularly more important for cancers, such as triple negative breast cancer, that currently lack targeted drugs.

DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Biallelic germline mutations in the SLC25A1 gene lead to combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a fatal systemic disease uniquely characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). How SLC25A1 deficiency contributes to D/L-2HGA and the role played by 2HG is unclear and no therapy exists. Both enantiomers act as oncometabolites, but their activities in normal tissues remain understudied. Here we show that mice lacking both SLC25A1 alleles exhibit developmental abnormalities that mirror human D/L-2HGA. SLC25A1 deficient cells undergo premature senescence, suggesting that loss of proliferative capacity underlies the pathogenesis of D/L-2HGA. Remarkably, D- and L-2HG directly induce senescence and treatment of zebrafish embryos with the combination of D- and L-2HG phenocopies SLC25A1 loss, leading to developmental abnormalities in an additive fashion relative to either enantiomer alone. Metabolic analyses further demonstrate that cells with dysfunctional SLC25A1 undergo mitochondrial respiratory deficit and remodeling of the metabolism and we propose several strategies to correct these defects. These results reveal for the first time pathogenic and growth suppressive activities of 2HG in the context of SLC25A1 deficiency and suggest that targeting the 2HG pathway may be beneficial for the treatment of D/L-2HGA.

Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs) in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense). The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms "mental retardation". To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus.

PRAJA, a RING-H2 E3 ligase, is abundantly expressed in brain tissues such as the cerebellum and frontal cortex, amongst others, and more specifically in neural progenitor cells as well as in multiple cancers that include glioblastomas. However, the specific role that Praja plays in neural development and gliomas remains unclear. In this investigation, we performed bioinformatic analyses to examine Praja1 and Praja2 expression across 29 cancer types, and observed raised levels of Praja1 and Praja2 in gliomas with an inverse relationship between Praja1 and apoptotic genes and Praja substrates such as Smad3. We analyzed the role of Praja in the developing brain through loss of function studies, using morpholinos targeting Praja1 in embryonic zebrafish, and observed that Praja1 is expressed prominently in regions enriched with neural precursor cell subtypes. Antisense Praja morpholinos resulted in multiple embryonic defects including delayed neural development likely through increased apoptosis. Further studies revealed high levels of Cdk1 with loss of Praja1 in TGF-β or insulin treated cells, supporting the link between Praja1 and cell cycle regulation. In summary, these studies underscore Praja's role in mammalian brain development and Praja1 deregulation may lead to gliomas possibly through the regulation of cell cycle and/or apoptosis.

Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.

The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported.

Environmental chemical (EC) exposures and our interactions with them has significantly increased in the recent decades. Toxicity associated biological characterization of these chemicals is challenging and inefficient, even with available high-throughput technologies. In this report, we describe a novel computational method for characterizing toxicity, associated biological perturbations and disease outcome, called the Chemo-Phenotypic Based Toxicity Measurement (CPTM). CPTM is used to quantify the EC "toxicity score" (Zts), which serves as a holistic metric of potential toxicity and disease outcome. CPTM quantitative toxicity is the measure of chemical features, biological phenotypic effects, and toxicokinetic properties of the ECs. For proof-of-concept, we subject ECs obtained from the Environmental Protection Agency's (EPA) database to the CPTM. We validated the CPTM toxicity predictions by correlating 'Zts' scores with known toxicity effects. We also confirmed the CPTM predictions with in-vitro, and in-vivo experiments. In in-vitro and zebrafish models, we showed that, mixtures of the motor oil and food additive 'Salpn' with endogenous nuclear receptor ligands such as Vitamin D3, dysregulated the nuclear receptors and key transcription pathways involved in Colorectal Cancer. Further, in a human patient derived cell organoid model, we found that a mixture of the widely used pesticides 'Tetramethrin' and 'Fenpropathrin' significantly impacts the population of patient derived pancreatic cancer cells and 3D organoid models to support rapid PDAC disease progression. The CPTM method is, to our knowledge, the first comprehensive toxico-physicochemical, and phenotypic bionetwork-based platform for efficient high-throughput screening of environmental chemical toxicity, mechanisms of action, and connection to disease outcomes.

RARRES1, a retinoic acid regulated carboxypeptidase inhibitor associated with fatty acid metabolism, stem cell differentiation and tumorigenesis is among the most commonly methylated loci in multiple cancers but has no known mechanism of action. Here we show that RARRES1 interaction with cytoplasmic carboxypeptidase 2 (CCP2) inhibits tubulin deglutamylation, which in turn regulates the mitochondrial voltage dependent anion channel (VDAC1), mitochondrial membrane potential, AMPK activation, energy balance and metabolically reprograms cells and zebrafish to a more energetic and anabolic phenotype. Depletion of RARRES1 also increases expression of stem cell markers, promotes anoikis, anchorage independent growth and insensitivity to multiple apoptotic stimuli. As depletion of CCP2 or inhibition of VDAC1 reverses the effects of RARRES1 depletion on energy balance and cell survival we conclude that RARRES1 modulation of CCP2-modulated tubulin-mitochondrial VDAC1 interactions is a fundamental regulator of cancer and stem cell metabolism and survival.

Dysregulation of the pathways that preserve mitochondrial integrity hallmarks many human diseases including diabetes, neurodegeration, aging and cancer. The mitochondrial citrate transporter gene, SLC25A1 or CIC, maps on chromosome 22q11.21, a region amplified in some tumors and deleted in developmental disorders known as velo-cardio-facial- and DiGeorge syndromes. We report here that in tumor cells CIC maintains mitochondrial integrity and bioenergetics, protects from mitochondrial damage and circumvents mitochondrial depletion via autophagy, hence promoting proliferation. CIC levels are increased in human cancers and its inhibition has anti-tumor activity, albeit with no toxicity on adult normal tissues. The knock-down of the CIC gene in zebrafish leads to mitochondria depletion and to proliferation defects that recapitulate features of human velo-cardio-facial syndrome, a phenotype rescued by blocking autophagy. Our findings reveal that CIC maintains mitochondrial homeostasis in metabolically active, high proliferating tissues and imply that this protein is a therapeutic target in cancer and likely, in other human diseases.

Transcriptional control of oxytocinergic cell development influences social, sexual, and appetite related behaviors and is implicated in disorders such as autism and Prader-Willi syndrome. Mediator 12 (Med12) is a transcriptional coactivator required for multiple facets of brain development including subsets of serotonergic and dopaminergic neurons. We surveyed hormone gene expression within the hypothalamo-pituitary axis of med12 mutant zebrafish embryos with a focus on oxytocin (oxt) expression. Some transcripts, such as oxt, vasopressin (avp) and corticotrophin releasing hormone (crh) are undetectable in the med12 mutant, while others are upregulated or downregulated to varying degrees. In med12 mutants, the expression patterns of upstream transcriptional regulators of oxytocinergic cell development remain largely intact in the pre-optic area, suggesting a more direct influence of Med12 on oxt expression. We show that Med12 is required for Wnt signaling in zebrafish. However, oxt expression is unaffected in Wnt-inhibited embryos indicating independence of Wnt signaling. In fact, overactive Wnt signaling inhibits oxt expression, and we identify a Wnt-sensitive period starting at 24 h post fertilization (hpf). Thus, Med12 and repression of Wnt signaling display critical but unrelated roles in regulating oxt expression.

Cancer metastasis is the primary cause of the high mortality rate among human cancers. Efforts to identify therapeutic agents targeting cancer metastasis frequently fail to demonstrate efficacy in clinical trials despite strong preclinical evidence. Until recently, most preclinical studies used mouse models to evaluate anti-metastatic agents. Mouse models are time-consuming and expensive. In addition, an important drawback is that mouse models inadequately model the early stages of metastasis which plausibly leads to the poor correlation with clinical outcomes.Here, we report an in vivo model based on xenografted zebrafish embryos where we select for progressively invasive subpopulations of MDA-MB-231 breast cancer cells. A subpopulation analogous to circulating tumor cells found in human cancers was selected by injection of MDA-MB-231 cells into the yolk sacs of 2 days post-fertilized zebrafish embryos and selecting cells that migrated to the tail. The selected subpopulation derived from MDA-MB-231 cells were increasingly invasive in zebrafish. Isolation of these subpopulations and propagation in vitro revealed morphological changes consistent with activation of an epithelial-mesenchymal transition program. Differential gene analysis and knockdown of genes identified gene-candidates (DDIT4, MT1X, CTSD, and SERPINE1) as potential targets for anti-metastasis therapeutics. Furthermore, RNA-splicing analysis reinforced the importance of BIRC5 splice variants in breast cancer metastasis. This is the first report using zebrafish to isolate and expand progressively invasive populations of human cancer cells. The model has potential applications in understanding the metastatic process, identification and/or development of therapeutics that specifically target metastatic cells and formulating personalized treatment strategies for individual cancer patients.