{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T01:36:45Z","timestamp":1773970605011,"version":"3.50.1"},"reference-count":55,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,8]],"date-time":"2023-01-08T00:00:00Z","timestamp":1673136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH\/NIDCR","doi-asserted-by":"publisher","award":["R35DE029086"],"award-info":[{"award-number":["R35DE029086"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Most human birth defects are phenotypically variable even when they share a common genetic basis. Our understanding of the mechanisms of this variation is limited, but they are thought to be due to complex gene-environment interactions. Loss of the transcription factor Gata3 associates with the highly variable human birth defects HDR syndrome and microsomia, and can lead to disruption of the neural crest-derived facial skeleton. We have demonstrated that zebrafish gata3 mutants model the variability seen in humans, with genetic background and candidate pathways modifying the resulting phenotype. In this study, we sought to use an unbiased bioinformatic approach to identify environmental modifiers of gata3 mutant craniofacial phenotypes. The LINCs L1000 dataset identifies chemicals that generate differential gene expression that either positively or negatively correlates with an input gene list. These chemicals are predicted to worsen or lessen the mutant phenotype, respectively. We performed RNA-seq on neural crest cells isolated from zebrafish across control, Gata3 loss-of-function, and Gata3 rescue groups. Differential expression analyses revealed 551 potential targets of gata3. We queried the LINCs database with the 100 most upregulated and 100 most downregulated genes. We tested the top eight available chemicals predicted to worsen the mutant phenotype and the top eight predicted to lessen the phenotype. Of these, we found that vinblastine, a microtubule inhibitor, and clofibric acid, a PPAR-alpha agonist, did indeed worsen the gata3 phenotype. The Topoisomerase II and RNA-pol II inhibitors daunorubicin and triptolide, respectively, lessened the phenotype. GO analysis identified Wnt signaling and RNA polymerase function as being enriched in our RNA-seq data, consistent with the mechanism of action of some of the chemicals. Our study illustrates multiple potential pathways for Gata3 function, and demonstrates a systematic, unbiased process to identify modifiers of genotype-phenotype correlations.<\/jats:p>","DOI":"10.3390\/ijms24021222","type":"journal-article","created":{"date-parts":[[2023,1,9]],"date-time":"2023-01-09T02:09:39Z","timestamp":1673230179000},"page":"1222","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Predicting Modifiers of Genotype-Phenotype Correlations in Craniofacial Development"],"prefix":"10.3390","volume":"24","author":[{"given":"Ranjeet D.","family":"Kar","sequence":"first","affiliation":[{"name":"Department of Molecular Biosciences, College of Natural Sciences, University of Texas at Austin, Austin, TX 78712, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johann K.","family":"Eberhart","sequence":"additional","affiliation":[{"name":"Department of Molecular Biosciences, College of Natural Sciences, University of Texas at Austin, Austin, TX 78712, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/j.cps.2013.12.003","article-title":"Classification, epidemiology, and genetics of orofacial clefts","volume":"41","author":"Watkins","year":"2014","journal-title":"Clin. Plast. Surg."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1002\/ajmg.a.33702","article-title":"Cranial neural crest cells on the move: Their roles in craniofacial development","volume":"155A","author":"Cordero","year":"2011","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1038\/nrg2933","article-title":"Cleft lip and palate: Understanding genetic and environmental influences","volume":"12","author":"Dixon","year":"2011","journal-title":"Nat. Rev. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1242\/dev.02281","article-title":"Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development","volume":"133","author":"Eberhart","year":"2006","journal-title":"Development"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3977","DOI":"10.1242\/dev.01943","article-title":"Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull","volume":"132","author":"Wada","year":"2005","journal-title":"Development"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3254","DOI":"10.1242\/dev.094938","article-title":"Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD","volume":"140","author":"McCarthy","year":"2013","journal-title":"Development"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1242\/dev.080473","article-title":"Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis","volume":"140","author":"Dougherty","year":"2013","journal-title":"Development"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1002\/dvdy.24566","article-title":"Zebrafish models of orofacial clefts","volume":"246","author":"Duncan","year":"2017","journal-title":"Dev. Dyn."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2204","DOI":"10.1002\/dvdy.22713","article-title":"Examination of a palatogenic gene program in zebrafish","volume":"240","author":"Swartz","year":"2011","journal-title":"Dev. Dyn."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1016\/j.mod.2006.07.007","article-title":"A role for GATA factors in Xenopus gastrulation movements","volume":"123","author":"Fletcher","year":"2006","journal-title":"Mech. Dev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1038\/ni.2692","article-title":"GATA-3 regulates the self-renewal of long-term hematopoietic stem cells","volume":"14","author":"Frelin","year":"2013","journal-title":"Nat. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1048","DOI":"10.1101\/gad.11.8.1048","article-title":"GATA4 transcription factor is required for ventral morphogenesis and heart tube formation","volume":"11","author":"Kuo","year":"1997","journal-title":"Genes Dev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1111\/j.1399-0004.2009.01170.x","article-title":"HDR (Hypoparathyroidism, Deafness, Renal dysplasia) syndrome associated to GATA3 gene duplication","volume":"76","author":"Bernardini","year":"2009","journal-title":"Clin. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1038\/35019088","article-title":"GATA3 haplo-insufficiency causes human HDR syndrome","volume":"406","author":"Groenen","year":"2000","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10605","DOI":"10.1038\/ncomms10605","article-title":"Genome-wide association study identifies multiple susceptibility loci for craniofacial microsomia","volume":"7","author":"Zhang","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1038\/76080","article-title":"Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system","volume":"25","author":"Lim","year":"2000","journal-title":"Nat. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Abe, M., Cox, T.C., Firulli, A.B., Kanai, S.M., Dahlka, J., Lim, K.C., Engel, J.D., and Clouthier, D.E. (2021). GATA3 is essential for separating patterning domains during facial morphogenesis. Development, 148.","DOI":"10.1242\/dev.199534"},{"key":"ref_18","first-page":"1285","article-title":"Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR)","volume":"6","author":"Swartz","year":"2013","journal-title":"Dis. Model. Mech."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1111\/j.1601-6343.2007.00391.x","article-title":"Review of the etiologic heterogeneity of the oculo-auriculo-vertebral spectrum (Hemifacial Microsomia)","volume":"10","author":"Hartsfield","year":"2007","journal-title":"Orthod. Craniofac. Res."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Swartz, M.E., Lovely, C.B., and Eberhart, J.K. (2021). Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling. PLoS Genet., 17.","DOI":"10.1371\/journal.pgen.1009579"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Barske, L., Askary, A., Zuniga, E., Balczerski, B., Bump, P., Nichols, J.T., and Crump, J.G. (2016). Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face. PLoS Genet., 12.","DOI":"10.1371\/journal.pgen.1005967"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1038\/75556","article-title":"Gene ontology: Tool for the unification of biology. The Gene Ontology Consortium","volume":"25","author":"Ashburner","year":"2000","journal-title":"Nat. Genet."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1437","DOI":"10.1016\/j.cell.2017.10.049","article-title":"A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles","volume":"171","author":"Subramanian","year":"2017","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.bpobgyn.2016.09.013","article-title":"Ovarian cancer in pregnancy","volume":"41","author":"Fruscio","year":"2017","journal-title":"Best Pract. Res. Clin. Obstet. Gynaecol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Luci, S., Giemsa, B., Hause, G., Kluge, H., and Eder, K. (2007). Clofibrate treatment in pigs: Effects on parameters critical with respect to peroxisome proliferator-induced hepatocarcinogenesis in rodents. BMC Pharmacol., 7.","DOI":"10.1186\/1471-2210-7-6"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/1476-511X-6-6","article-title":"Feeding oxidized fat during pregnancy up-regulates expression of PPARalpha-responsive genes in the liver of rat fetuses","volume":"6","author":"Ringseis","year":"2007","journal-title":"Lipids Health Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1038\/ng0995-40","article-title":"Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis","volume":"11","author":"Pandolfi","year":"1995","journal-title":"Nat. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1083\/jcb.200309096","article-title":"A divergent canonical WNT-signaling pathway regulates microtubule dynamics: Dishevelled signals locally to stabilize microtubules","volume":"164","author":"Ciani","year":"2004","journal-title":"J. Cell Biol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Fukuda, Y., Sano, O., Kazetani, K., Yamamoto, K., Iwata, H., and Matsui, J. (2016). Tubulin is a molecular target of the Wnt-activating chemical probe. BMC Biochem., 17.","DOI":"10.1186\/s12858-016-0066-9"},{"key":"ref_30","first-page":"2741","article-title":"Comparison of the effects of vinblastine, vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation in vitro","volume":"45","author":"Jordan","year":"1985","journal-title":"Cancer Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1016\/j.biopha.2018.05.088","article-title":"Broad targeting of triptolide to resistance and sensitization for cancer therapy","volume":"104","author":"Hou","year":"2018","journal-title":"Biomed. Pharmacother."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1817","DOI":"10.1016\/j.athoracsur.2015.05.074","article-title":"Triptolide Inhibits Lung Cancer Cell Migration, Invasion, and Metastasis","volume":"100","author":"Reno","year":"2015","journal-title":"Ann. Thorac. Surg."},{"key":"ref_33","first-page":"149","article-title":"Immunosuppressive, anti-inflammatory and anti-cancer properties of triptolide: A mini review","volume":"6","author":"Ziaei","year":"2016","journal-title":"Avicenna J. Phytomed."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2780","DOI":"10.1158\/1535-7163.MCT-09-0549","article-title":"Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA","volume":"8","author":"Vispe","year":"2009","journal-title":"Mol. Cancer Ther."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1002\/mpo.2950070405","article-title":"Acute leukemia in pregnancy: Transient neonatal myelosuppression after combination chemotherapy in the mother","volume":"7","author":"Okun","year":"1979","journal-title":"Med. Pediatr. Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Al-Aamri, H.M., Ku, H., Irving, H.R., Tucci, J., Meehan-Andrews, T., and Bradley, C. (2019). Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia. BMC Cancer, 19.","DOI":"10.1186\/s12885-019-5377-y"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1002\/jat.3007","article-title":"Cardiotoxicity evaluation of anthracyclines in zebrafish (Danio rerio)","volume":"35","author":"Han","year":"2015","journal-title":"J. Appl. Toxicol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1089\/dna.1.1988.7.483","article-title":"Effects of antibiotics on RNA polymerase III transcription","volume":"7","author":"Logan","year":"1988","journal-title":"DNA"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Pallis, M., Burrows, F., Whittall, A., Boddy, N., Seedhouse, C., and Russell, N. (2013). Efficacy of RNA polymerase II inhibitors in targeting dormant leukaemia cells. BMC Pharmacol. Toxicol., 14.","DOI":"10.1186\/2050-6511-14-32"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1186\/s13046-019-1232-x","article-title":"Inhibition of RNA polymerase III transcription by Triptolide attenuates colorectal tumorigenesis","volume":"38","author":"Liang","year":"2019","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1089\/zeb.2008.0563","article-title":"The emerging role of Wnt\/PCP signaling in organ formation","volume":"6","author":"Dale","year":"2009","journal-title":"Zebrafish"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/ncb716","article-title":"The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos","volume":"4","author":"Park","year":"2002","journal-title":"Nat. Cell Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"255","DOI":"10.4161\/org.7.4.18797","article-title":"Planar cell polarity signaling in craniofacial development","volume":"7","author":"Topczewski","year":"2011","journal-title":"Organogenesis"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1016\/j.ydbio.2013.06.012","article-title":"Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis","volume":"381","author":"Kamel","year":"2013","journal-title":"Dev. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"13730","DOI":"10.1074\/jbc.M116.733766","article-title":"Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation","volume":"291","author":"Brinkmann","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1002\/aja.1002030302","article-title":"Stages of embryonic development of the zebrafish","volume":"203","author":"Kimmel","year":"1995","journal-title":"Dev. Dyn."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1006\/dbio.2002.0711","article-title":"In vivo imaging of embryonic vascular development using transgenic zebrafish","volume":"248","author":"Lawson","year":"2002","journal-title":"Dev. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/nn2025","article-title":"CNS-derived glia ensheath peripheral nerves and mediate motor root development","volume":"11","author":"Kucenas","year":"2008","journal-title":"Nat. Neurosci."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2713","DOI":"10.1242\/dev.048470","article-title":"Regulatory interactions specifying Kolmer-Agduhr interneurons","volume":"137","author":"Yang","year":"2010","journal-title":"Development"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"R36","DOI":"10.1186\/gb-2013-14-4-r36","article-title":"TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions","volume":"14","author":"Kim","year":"2013","journal-title":"Genome Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"3","DOI":"10.14806\/ej.17.1.200","article-title":"Cutadapt removes adapter sequences from high-throughput sequencing reads","volume":"17","author":"Martin","year":"2011","journal-title":"EMBnet J."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"D754","DOI":"10.1093\/nar\/gkx1098","article-title":"Ensembl 2018","volume":"46","author":"Zerbino","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1089\/omi.2011.0118","article-title":"clusterProfiler: An R package for comparing biological themes among gene clusters","volume":"16","author":"Yu","year":"2012","journal-title":"OMICS"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1080\/10520290701333558","article-title":"A two-color acid-free cartilage and bone stain for zebrafish larvae","volume":"82","author":"Walker","year":"2007","journal-title":"Biotech. Histochem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/2\/1222\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:03:10Z","timestamp":1760119390000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/2\/1222"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,8]]},"references-count":55,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["ijms24021222"],"URL":"https:\/\/doi.org\/10.3390\/ijms24021222","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,8]]}}}