microinjection-crisprcas9-protein-into-channel-catfish-ictalurus

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, <em>Ictalurus punctatus</em>, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

University of Michigan

The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate.

Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish.

We conducted a genome-wide association study (GWAS) of anorexia nervosa (AN) using a stringently defined phenotype. Analysis of phenotypic variability led to the identification of a specific genetic risk factor that approached genome-wide significance (rs929626 in EBF1 (Early B-Cell Factor 1); P = 2.04 × 10(-7); OR = 0.7; 95% confidence interval (CI) = 0.61-0.8) with independent replication (P = 0.04), suggesting a variant-mediated dysregulation of leptin signaling may play a role in AN. Multiple SNPs in LD with the variant support the nominal association. This demonstrates that although the clinical and etiologic heterogeneity of AN is universally recognized, further careful sub-typing of cases may provide more precise genomic signals. In this study, through a refinement of the phenotype spectrum of AN, we present a replicable GWAS signal that is nominally associated with AN, highlighting a potentially important candidate locus for further investigation.

A genome-wide association study of anorexia nervosa suggests a risk locus implicated in dysregulated leptin signaling.

Cetaceans, a group of mammals adapted to the aquatic environment that descended from terrestrial artiodactyls, exhibit tremendous interspecific differences in a number of phenotypes, including feeding behavior, such as filter feeding in the Mysticeti vs prey-hunting Odontoceti, and size, with the smallest cetacean, the vaquita, at 1.4 meters and the largest, the blue whale, reaching 33 meters. The Melanocortin-4 receptor (MC4R) regulates food intake, energy balance, and somatic growth in both mammals and teleosts. In this study, we examined allelic variants of the MC4R in cetaceans. We sequenced the MC4R from 20 cetaceans, and pharmacologically characterized 17 of these protein products. Results identified a single variation at amino acid 156 in the MC4R from representative species of major cetacean lineages uniquely associated with the toothed whales or Odontoceti (arginine at 156) and baleen whales or Mysticeti (glutamine at 156). The Q156 receptor variant found in the larger baleen whales was functionally less responsive to its endogenous anorexigenic ligand, α-MSH. Furthermore, the R156 receptor variant showed greater constitutive activity and a higher affinity for ligand. These data suggest that the MC4R may be one gene involved in the evolution of feeding ecology, energy balance, and body size in cetaceans.

Functional variants of the melanocortin-4 receptor associated with the Odontoceti and Mysticeti suborders of cetaceans.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Erratum: A genome-wide association study of anorexia nervosa suggests a risk locus implicated in dysregulated leptin signaling.

Haploinsufficiency of the melanocortin-4 receptor, the most common monogenetic obesity syndrome in humans, is associated with a reduction in autonomic tone, bradycardia, and incidence of obesity-associated hypertension. Thus, it has been assumed that melanocortin obesity syndrome may be protective with respect to obesity-associated cardiovascular disease. We show here that absence of the melanocortin-4 receptor (MC4R) in mice causes dilated cardiomyopathy, characterized by reduced contractility and increased left ventricular diameter. This cardiomyopathy is independent of obesity as weight matched diet induced obese mice do not display systolic dysfunction. Mc4r cardiomyopathy is characterized by ultrastructural changes in mitochondrial morphology and cardiomyocyte disorganization. Remarkably, testing of myocardial tissue from Mc4r-/- mice exhibited increased ADP stimulated respiratory capacity. However, this increase in respiration correlates with increased reactive oxygen species production - a canonical mediator of tissue damage. Together this study identifies MC4R deletion as a novel and potentially clinically important cause of heart failure.

Loss of the melanocortin-4 receptor in mice causes dilated cardiomyopathy.

Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1-5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency.

Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency.

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gαs-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gαs and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl+ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl+ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.

Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay.

Subjects spending much time sitting have increased risk of obesity but the mechanism for the antiobesity effect of standing is unknown. We hypothesized that there is a homeostatic regulation of body weight. We demonstrate that increased loading of rodents, achieved using capsules with different weights implanted in the abdomen or s.c. on the back, reversibly decreases the biological body weight via reduced food intake. Importantly, loading relieves diet-induced obesity and improves glucose tolerance. The identified homeostat for body weight regulates body fat mass independently of fat-derived leptin, revealing two independent negative feedback systems for fat mass regulation. It is known that osteocytes can sense changes in bone strain. In this study, the body weight-reducing effect of increased loading was lost in mice depleted of osteocytes. We propose that increased body weight activates a sensor dependent on osteocytes of the weight-bearing bones. This induces an afferent signal, which reduces body weight. These findings demonstrate a leptin-independent body weight homeostat ("gravitostat") that regulates fat mass.

Article	Year
Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish. Scientific reports\| PubMed ID: 28317906	2017
A genome-wide association study of anorexia nervosa suggests a risk locus implicated in dysregulated leptin signaling. Scientific reports\| PubMed ID: 28630421	2017
Functional variants of the melanocortin-4 receptor associated with the Odontoceti and Mysticeti suborders of cetaceans. Scientific reports\| PubMed ID: 28720755	2017
Erratum: A genome-wide association study of anorexia nervosa suggests a risk locus implicated in dysregulated leptin signaling. Scientific reports\| PubMed ID: 28827695	2017
Loss of the melanocortin-4 receptor in mice causes dilated cardiomyopathy. eLife\| PubMed ID: 28829041	2017
Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency. Molecular metabolism\| PubMed ID: 29031731	2017
Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29058194	2018
Body weight homeostat that regulates fat mass independently of leptin in rats and mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29279372	2018

Roger Cone

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