Ela W. Knapik, M.D.
Associate Professor, Cell and Developmental Biology
Protein Secretion, and Extracellular Matrix, Regulation of Cell Shape Changes in Morphogenesis, Modeling Human Developmental and and Neuropsychiatric Disorders, DNA Biobank (BioVU), Regulation of Cell Shape Changes in in Morphogenesis
M.D., Jagiellonian University, Cracow, Poland
Postdoctoral, Colorado State University, Fort Collins
Postdoctoral, Massachusetts General Hospital and Harvard Medical School, Boston, MA
We have focused our investigation research oin three main areas:
(i)The mechanisms and pathways that directing extracellular matrix traffic and deposition
(i)The role of protein trafficking in chondrocyte maturation and cell shape changes during morphogenesis
(ii)The pathophysiology of human diseasess, including developmental diseases and neuropsychiatric disorders
(iii)Modeling of human variants for drug development and testing
The Knapik laboratory uses in vivo approaches in Our laboratory is interested in understanding mechanisms and pathways that direct neural crest stem cells, and mediate their differentiation into various derivatives. We Our main model system, zebrafish (Danio rerio) because this model system lends itselfis superbly suited for genetic and developmental analyseis by being because of abundant availability of transparent embryos developing outside the mother that are accessible for manipulation and direct live observation. We use genetic mutants induced by CRISPR genome editing or chemically induced random variants to assess developmental phenotypes, gene function and pathway interactions. This small fish can be used in a mutagenesis screen and its genome is amenable to efficient positional cloning of mutations and phenotypic analysis. In parallel, we use mammalian cells in culture and biochemical approaches to address our research interests.
(iI) use a particularly effective approach to dissect the complex processes of cell specification and differentiation namely a combination of embryological and genetic methods and ask how gene networks control cell behavior and cell fate.
The first steps en route to answer our research these questions, beginning with individual genes, is to develop and characterize mutations that produce specific neural crest phenotypes. , Using a forward genetic screens and mutational analysis, we have identified point mutations responsible for craniofacial malformations in zebrafish, and we are able to use these established mutant zebrafish lines to examine how defects in the cellular secretory pathway affects chondrocyte cell shape and extracellular matrix deposition. Disruptions in the synthesis, processing or secretion of extracellular matrix (ECM) extracellular matrix macromolecules are associated with human diseases, including osteogenesis imperfectaimperfecta (OI), multiple epiphyseal dysplasia, joint dislocations, cranio-lenticulo-sutural dysplasia, and congenital disorders of glycosylation (CDG). Our laboratory is able to adequatelyhas successfully modeled these human diseases in zebrafish and contributed to understanding of basic in order to better basic developmental mechanisms and cellular pathways that direct skeletal development and homeostasisECM traffic and deposition. Using phenotype-driven forward genetic screens and positional cloning, we have identified point mutations responsible for skeletal malformations in zebrafish,; and currently, we are working to show how defects in the secretory pathway affect skeletal biology, chondrocyte cell shape and ECM deposition.
(IIii) Additionally, Concurrently, we are investigating modeling other human diseases, including developmentalMendelian diseasedisorders such as cChylomicron rRetention dDisease and zZinc tTransporter deficits, as well as and comorbidities associated with complex neuropsychiatric disordersdiseases (ASD, MDD, SCZ, BD, ADHD, OCD), such as skeletal dysmorphology, seizures and gastrointestinal disturbances in zebrafish. In collaboration with the Vanderbilt Genetics DepartmentInstitute (VGI) investigators,, we examine effects of genetic variation, identified by statistical genetic methods patient data stored in BioVU, on gene function and resulting phenotypic spectrum. , Vanderbilt’s collection of de-identified DNA samples, to identify potential gene candidates associated with a human disease. Using CRISPR/Cas9 genome editing,, we are currently producinggenerating zebrafish mutantknockout and transgenic lines that are evaluated by in vivo and ex vivo methods for discovery of primary genetic mechanisms underlying the pathophysiology of these diseases. with single nucleotide polymorphisms (SNPs) on genes identified through BioVU. Our goal is to use genetic editing and mutational analysis to better understand the pathophysiology of human disease that have alluded scientists and clinicians for years. Moreover, we are able to use the skeletal biology of zebrafish in combination with SNP modeling to investigate comorbidities that are associated with neuropsychiatric disorders, including craniofacial dysmorphologies. establish gene hierarchy and cooperation among regulators that will control neural crest progenitors specification and differentiation. Neural crest stem cell genes and the genetic mutations are our primary tools to study gene expression, regulation, epistasis, cell fate, behavior, proliferation and survival.
Our main model system, zebrafish (Danio rerio) lends itself superbly to genetic and developmental analysis because of abundant availability of transparent embryos developing outside the mother that are accessible to manipulation and direct observation. This small fish can be used in a mutagenesis screen and its genome is amenable to efficient positional cloning of mutations and phenotypic analysis. In parallel, we use mammalian cells in culture and biochemical approaches to address our questions.
We have focused our investigation on two areas:
(i) the genetic network of transcription factors orchestrating transition of neural crest stem cell to derivatives like craniofacial cartilage,
(ii) and the role of cellular secretory pathway in chondrocyte maturation.
We use positional cloning approaches to help us identify point mutations responsible for craniofacial malformations. We have already found a set of factors critical for neural crest specification in the mutations mont blanc, mother superior and quadro. Now, we are dissecting the genetic interactions among these factors and further search for novel components of the genetic network.
Concurrently, we are investigating a phenotypic series of mutations affecting chondrocyte differentiation. Chondrocytes originate from neural crest stem cells and constitute most of the larval head skeleton. Surprisingly, we have discovered that the mutations in crusher and bulldog loci disrupt genes in the cellular secretory pathway, while the stumpf locus is critical in posttranslational protein modification and folding. These results open a new vista for research on the role of secretory pathway in a global context of embryonic morphogenesis and disease.
(IIIiii) Zebrafish is ideally suited for modeling of human single nucleotide polymorphisms (SNPs) in genes identified through BioVU variant investigations. We can test whetweather a specific SNP increases or decreases gene activity, and thus alterings protein function, such as for example a receptor activity, for example. These models are particularly helpful in testing responses to known pharmacological agents and in drug screens for new pharmaceuticals. We are using CRISPR/Cas9 –based knockout and knockin strategiesy in stable zebrafish lines that are then evaluated for phenotype and levels of gene function. This early functional genomics assessment model offers rapid and powerful tool in drug discovery pipeline.
Hockman D., Burns AJ, Schlosser G, Gates KP, Jevans B, Mongera A, Fisher S, Unlu G, Knapik EW, Kaufman CK, Mosimann C, Zon LI, Lancman JJ, Dong PDS, Lickert H, Tucker AS and Baker CVH. (2017) Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes. eLife; PMID: 28387645
Hockman D, Burns AJ, Schlosser G, Gates KP, Jevans B, Mongera A, Fisher S, Unlu G, Knapik EW, Kaufman CK, Moismann C, Zon LI, Lancman JJ, Dong PDS, Lickert H, Tucker AS, Baker CVH. Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes. Elife. 2017 Apr 7;6. pii: e21231. doi: 10.7554/eLife.21231, PMID: 28387645, PMCID: PMC5438250
Luderman LN, Unlu G, Knapik EW. (2017) Zebrafish Developmental Models of Skeletal Diseases. Curr Top Dev Biol. Epub 2017 Jan 4; 124: 81-124. PMID: 28335865 DOI: 10.1016/bs.ctdb.2016.11.004
Levic DS, Minkel JR, Wang WD, Rybski WM, Melville DB, Knapik EW (2015) Animal model of Sar1b deficiency presents lipid absorption deficient similar to Anderson disease. J Mol. Med (Berl) 93:165-76. PMCID: PMC4319984
Unlu G, Levic DS, Melville DB, Knapik EW. (2014) Trafficking mechanisms of extracellular matrix molecules: insights from vertebrate development and human diseases. Int J Biochem Cell Biol. 2014 Feb; 47:57-67. PMID: 24333299. Doi: 10.1016/j.biocel.2013.11.005.
Wang WD, Melville DB, Monero-Balaguer M, Hatzopoulous AK, Knapik EW. Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population. Dev Biol. 2011 Dec 1; 360(1): 173-85. PMID: 21963426. DOI: 10.1016/j.ydbio.2011.09.019.
Zou P, Wu SY, Koteiche HA, Mishra S, Levic DS, Knapik E, Chen W, Mchaourab HS. A conserved role of aA-crystallin in the development of the zebrafish embryonic lens. Exp. Eye Res [print-electronic]. 2015 Sep; 138: 104-13. PMID: 26149094, PMCID: PMC4638411, PII: S0014-4835(15)00220-1, DOI: 10.1016/j.exer.2015.07.001, ISSN: 1096-0007.
Müller II, Melville DB, Tanwar V, Rybski W, Mukherjee A, Shoemaker BM, Wang WD, Schoenhard JA, Roden DM, Darbar D, Knapik EW, Hatzopoulos AK (2013) Functional modeling in zebrafish demonstrates that the atrial-fibrillation-associated gene GREM2 regulates cardiac laterality, cardiomyocyte differentiation and atrial rhythm. Dis Model Mech. 6: 332-341, PMID: 23223679Levic DS, Minkel JR, Wang WD, Rybski WM, Melville DB, Knapik EW. Animal model of Sar1b deficiency presents lipid absorption deficits similar to Anderson disease. J. Mol. Med [print-electronic]. 2015 Feb; 93(2): 165-76. PMID: 25559265, PMCID: PMC4319984, DOI: 10.1007/s00109-014-1247-x, ISSN: 1432-1440.
Levic DS, Minkel JR, Wang WD, Rybski WM, Melville DB, Knapik EW. Animal model of Sar1b deficiency presents lipid absorption deficits similar to Anderson disease. J. Mol. Med [print-electronic]. 2015 Feb; 93(2): 165-76. PMID: 25559265, PMCID: PMC4319984, DOI: 10.1007/s00109-014-1247-x, ISSN: 1432-1440.
Melville DB, Monero-Balaguer M, Levic DS, Bradley K, Smith JR, Hatzopoulous AK, Knapik EW. (2011) The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrixECM proteins in skeletal morphogenesis. Dis Model Mech. 2011 Nov; 4(6):763-76. PMID: 21729877. Doi: 10.1242/dmm.007625.
Wang WD. Montero-Balaguer M. Hatzopoulos A.K. and E.W. Knapik (2011) Tfap2a and Foxd3 synergistically regulate early steps in development of the neural crest progenitor population. Dev Biol., 360: 173-85, PMID: 21963426,
Sarmah S, Barrallo-Gimeno A, Melville DB, Topczewski J, Solinca-Krezel L, Knapik EW. (2010) Sec24D-dependent transport of extracellular matrix proteins is required for zebrafish skeletal morphogenesis. PLoS One. 2010 Apr 28; 5(4):e10367. PMID: 20442775. Doi: 10.1371/journal.pone.0010367.
Lang MR, Lapierre LA, Goldenring JR, Frotscher M, and EW. Knapik (2006) Secretory