From 8/18/2017 through 9/7/2018 Feldman engaged in research projects with seven labs. Porter Lab (NICHD): Genetic Dissection and Creation of Human Disease Models of Sterol Metabolism. Niemann-Pick disease type C is a rare autosomal recessive disease caused by the accumulation of cholesterol and glycolipids in late endosomes/lysosomes. A phenotypic characterization and proof of principle drug-screening approach was published in 2018 for zebrafish carrying mutant alleles of npc1, the zebrafish ortholog to the human Niemann-Pick Type C disease gene NPC1, with one of these alleles having been generated by the Core in previous years. In June of 2018, the Core received supplemental Directors Award funding to compare precise genome editing methods and efficiencies, with the creation of two new zebrafish npc1 alleles to model homologous disease-causing alleles in humans and potentially use to test drug therapies as part of the scope. The Core has previously used CRISPR-Cas9 technology to create additional genetic mutant lines for the Porter lab in genes with roles in other steps of cholesterol metabolism, such as the zebrafish ortholog to the human Smith-Lemli-Opitz Syndrome gene, dhcr7. Phenotypic characterization of these mutant lines by the Porter lab is ongoing. Stratakis Lab (NICHD): Function of Zebrafish Orthologs to Human Genes Implicated in Disorders of the Pituitary-Adrenal Axis. (1) Gigantism arises due to excess growth hormone (GH) secretion during childhood, before the growth plates close. Since 2012, the Core has supported this labs investigation of the zebrafish ortholog to a human gene implicated as a driver of gigantism. The Core used CRISPR-Cas9 methods in previous years to generate zebrafish carrying loss-of-function mutations in four zebrafish orthologs to human genes implicated by the Stratakis lab in human growth anomalies. Characterization of the resulting phenotypes is ongoing. (2) The Core also continues to support the Stratakis labs investigation into the function of two zebrafish orthologs to human adrenal hyperplasia and Cushing disease-associated genes, through the study of mutant lines that the Core previously helped to generate (one) or acquire (one) Phenotypic characterization has found notable effects on juvenile growth in the case of one gene and on early embryogenesis in the case of the other. Phenotypic characterization of these lines will continue into 2018-19. Last year we generated mutants for six new genes whose human orthologs are implicated in adrenal hyperplasia and/or Cushing disease and phenotypic characterization of these mutations has begun. Kaler Lab (NICHD): Modeling Copper Deficiency-Associated Distal Motoneuropathy. ATP7A is a copper-binding ATPase. Complete loss of ATP7A causes a severe human disease leading to childhood death. Two ATP7A missense mutations cause a milder syndrome distal motoneuropathy that is nevertheless debilitating to children and young adults. Since 2013, the Core has supported a project to clarify the structure-function relationship of ATP7A and motor neuron defects from the perspective of these missense mutations. Using CRISPR-Cas9 technology in combination with donor DNA the core successfully created a zebrafish cognate to one of these alleles in early 2018. Initial phenotype analysis indicates an intermediate phenotype characterized by hypopigmentation, but no obvious diminishment of motor function within the first four months of life. The Directors Award scope includes creation of the second Atp7a allele, and preliminary results indicate a degree of success. Marini Lab (NICHD). Function of Osteogenesis Imperfecta (OI) genes. The Directors Award scope includes creation of an amino-acid substitution in the zebrafish ifitm5 gene that will be a cognate to the human theIFITM5S40L substitution known to cause type VI spectrum OI that is normally associated with mutations in PEDF. This project aims to provide an inroad to understanding the potential link between IFTM5 and PEDF functions. Blackshear lab (NIEHS): Assessing Functions of a Zinc-Finger Protein Gene Family in Zebrafish. The Blackshear lab is interested in dissecting the connections of a family of zinc-finger proteins and blood development. They contracted the NICHD Zebrafish Core to create null mutations in seven zebrafish orthologues and assist with preliminary phenotype characterization. Last year we created null alleles for six out of the seven genes, and alleles for the seventh were obtained this year. Phenotypes for all alleles tested have proven to be weak or absent and the Blackshear lab is now comparing the transcriptomes of mutants versus control siblings in case subtle molecular consequences of these gene disruptions can illuminate their function. Meilleur lab (NINR): Testing the Ability of Small Molecules to Mitigate Myopathy in Zebrafish ryr1b Mutants. Dr. Meilleur and colleagues began this year to test candidate drugs for their ability to potentially ameliorate muscle defects seen in zebrafish mutants that carry mutations in a gene, ryr1b, for which mutations in its human counterpart are implicated in various myopathies. Last year we acquired larvae carrying the ryr1b mutation from an outside source and have raised them to adulthood. This year we developed a behavioral assay using the Viewpoint Zebrabox system that can identify ryr1b homozygous mutant larvae by virtue of their decreased movement over time. Three drugs were tested for their ability to ameliorate this movement deficit, establishing proof-of-principle confirmation that this system can be used to screen candidate drugs. We have also developed strategies for pre-selecting equal numbers of WT and mutant siblings in advance of these assays, thereby increasing the efficiency of the screening pipeline. Tuchman Lab (Childrens National Medical Center): Finding Neuroprotective Drugs to Mitigate Hyperammonemia, a Consequence of Urea Cycle Defects & Liver Failure. Exposure of the brain to high ammonia causes neurcognitive deficits, intellectual disabilities, coma and death. Since 2012, the Core has helped this lab to use zebrafish embryos to identify small molecules able to diminish the effects of hyperammonemia. In the initial few years, a library of hundreds of small molecules with known safety profiles for humans was screened and several promising candidates were identified for follow-up validation studies in zebrafish and other animal models. A manuscript summarizing this work is currently being drafted. Over the last two years, the Core has supported a re-implementation of this screen using a larger library of 10,000 compounds, bolstered by additional personnel from the Tuchman lab, and the Cores implementation of NICHDs massive embryo production systems as a source for embryos. Additional candidate compounds were thus identified and ongoing secondary screens and dose-response studies on lead compounds will continue in 2018-19.
| Tseng, Wei-Chia; Loeb, Hannah E; Pei, Wuhong et al. (2018) Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds. Dis Model Mech 11: |
| Gore, Aniket V; Athans, Brett; Iben, James R et al. (2016) Epigenetic regulation of hematopoiesis by DNA methylation. Elife 5:e11813 |
| Trivellin, Giampaolo; Bjelobaba, Ivana; Daly, Adrian F et al. (2016) Characterization of GPR101 transcript structure and expression patterns. J Mol Endocrinol 57:97-111 |
| Feldman, B; Tuchman, M; Caldovic, L (2014) A zebrafish model of hyperammonemia. Mol Genet Metab 113:142-7 |
| Roessler, Erich; Hu, Ping; Hong, Sung-Kook et al. (2012) Unique alterations of an ultraconserved non-coding element in the 3'UTR of ZIC2 in holoprosencephaly. PLoS One 7:e39026 |
