Research Focus - 2020 to 2024

Lab Projects

Master
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The Role of Planar Cell Polarity Genes in Complex Birth Defects

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The planar cell polarity (PCP) genes have recently been associated with increased risks of both neural tube and craniofacial malformations. In humans, polymorphisms in the PCP gene VANGL1 were found in several families affected by spina bifida, while VANGL2 SNPs were found more frequently in anencephalic fetuses. The primary challenge remains to identify which gene variants under what circumstances increase NTD risk to developing embryos. PCP genes are also known to be mutated in several mouse models of NTDs, including the Loop tail (Lp) mouse, the Circle tail (Crc) mouse mutation, and the Celsr gene that is mutated in the Crash (Crsh) mouse. The Finnell Laboratory has developed a mouse line lacking the PCP gene, Fuzzy (Fuz), whose phenotype, which include NTDs, suggests an interaction with the Wnt/Shh pathways. In addition to NTDs, the Wnt/Shh pathways have been implicated in craniofacial defects. The development of the vertebrate craniofacies requires complicated tissue-tissue interactions between all germ layers and coordinated movements in three dimensions.

Small variations in programming the morphogenetic events can lead to a diverse range of congenital defects. A majority of all human birth defects are associated with some form of craniofacial malformation, creating tremendous medical and social burdens, as many require lifelong care. Fuz is essential for craniofacial development and normal closure of the cranial sutures. The Fuz knockout mouse presents with craniofacial deformities including: hypoplastic mandible, missing incisors, malformed molars, hyperplastic Meckel’s cartilage, premature closure of the sutures, anophthalmia and missing tongue.

Given the critical function of canonical Wnt signaling in craniofacial development, we hypothesize that canonical Wnt signaling will be increased in the Fuz-/- mutant mice. The sonic hedgehog (Shh) signaling pathway has also been shown to work through primary cilia. Thus, we will also test the hypothesis that Fuz inactivation affects Shh signaling and the expression of Shh downstream genes during craniofacial development. We will determine how Fuz regulates target gene expression through both the Wnt and Shh signaling pathways and its impact on birth defects such as craniosynostosis.

These studies are supported by NIH grant R01HD093758-01 The Role of GPR161 in the Etiology of Neural Tube Defects. Funding period: Sept. 9, 2018 - Aug. 31, 2023

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Using Embryonic Stem Cells to Screen for Reproductive Toxicants

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There are over 85,000 industrial chemicals presently registered with the federal government, the vast majority are inadequately studied with regards to their potential environmental and biological impacts. Over 30,000 of these industrial chemicals are sold at quantities exceeding 400 million tons per year; however, there also remains a considerable knowledge gap regarding the relationship between exposure to these industrial chemicals and possible adverse health effects. Many of these chemicals are released into the environment and can be absorbed via ingestion, inhalation, or dermal exposures. Additionally, these chemicals can also be transported to the fetus after maternal exposure and can have adverse effects on fetal and neonatal health. This lack of information on industrial chemicals released into the environment leaves the federal government with an ever-increasing number of chemicals that may contribute to immediate and long-term health effects in exposed animal and human populations, most of which need to be characterized and prioritized by their individual health risks. The Finnell Laboratory is working to develop a solution to this problem via high-throughput, high-information content biological screens on industrial chemicals. We hypothesize that high throughput screening (HTS) ESCs can recapitulate observations from in vivo models exposed to environmental pollutants. Further, in the absence of a priori toxicological data, we hypothesize that HTS of ESC can be used for prediction of in vivo impact, risk assessment, and prioritizing of chemicals for in vivo testing and epidemiological studies.

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How Does Periconceptional Folic Acid Prevent Birth Defects?

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Birth defects are among the leading pediatric healthcare issues, yet there are few prevention strategies and the prevalence of birth defects in the U.S. has remained relatively stable for decades, despite folic acid fortification of the food supply.  Neural tube defects (NTDs) are caused by failures in embryonic central nervous system development and include posterior defects, e.g. spina bifida, anterior defects, e.g. anencephaly, or disruption along the entire neural tube i.e. craniorachischisis. Fetuses with anencephaly and craniorachischisis die in utero or are stillborn. Most spina bifida patients can survive, yet are likely to have severe, life-long disabilities, and are at risk for psychosocial maladjustment. Globally, these defects are estimated to affect approximately 18.6 per 10,000 live births, and the prevalence of NTDs is 1–2 per 1,000 births in most regions of the US. There are approximately 2,300 NTD-affected pregnancies in the US each year, whose lifetime medical costs are estimated to be $560,000 per child or $1.68 billion per year nationwide. Approximately 30% of NTDs cannot be prevented by maternal periconceptional folate supplementation. Our preliminary data showed that rare mutations in PCP pathway could increase the risk of NTDs, while mutagens such as polycyclic aromatic hydrocarbons  were enriched in the placentas of NTDs fetuses/infants. We hypothesized that NTDs are caused by combinations of rare multiple mutations. Higher mutation rates result in a higher NTDs prevalence. It is possible that folic acid can prevent NTDs by reducing the mutation rate. Furthermore, we hypothesize that paternal supplementation with folic acid can further reduce NTD rates. We have a unique opportunity to evaluate folate supplement effect on mutation rate by leveraging previously collected biological samples from NTD patients and controls from several distinct cohorts. Additionally, we propose to use mouse models to better understand the relationship between folate and mutation rates in vivo. The results of this study will provide an explanation of how folate prevents NTDs and will reveal novel pathways for intervention on folate resistant NTDs. Hence, the successful completion of the proposed studies is likely to have substantial impact on our understanding of conditions that affect ~6% of births worldwide and constitute one of the major healthcare concerns for the youngest members of society.


This work is supported by NIH grant R01HD081216 Folic Acid, Parental Mutation Rates, and the Risk for Neural Tube Defects. Funding Period: 8/1/2015-7/31/2020.
 

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How to develop intervention strategies for non-folate responsive neural tube defects

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Neural tube defects (NTDs) are among the most common birth defects in humans. The causes of NTDs are believed to be multifactorial, including genetic, environmental, and nutritional factors. Maternal folic acid (FA) status is one of the strongest links to NTD susceptibility. Numerous studies have shown that supplemental FA can reduce NTD prevalence by as much as 70% in some populations. Despite more than 40 years of intensive effort, we still do not understand the mechanisms that underlie these folate-dependent processes. We have begun to address this existing data gap utilizing a new mouse NTD model (Mthfd1l KO) that closely replicates the human NTD phenotype and does not require additional nutritional intervention to express the NTD phenotype. In this new mouse model, loss of a specific folate-dependent enzyme (mitochondrial MTHFD1L) leads to NTDs. This is the most specific metabolic defect yet associated with NTD susceptibility/etiology and suggests that FA provides essential one-carbon units for nucleotide and methyl group biosynthesis. These biosynthetic pathways are especially active in the rapidly growing embryo, where they supply the nucleotides and methyl groups required for cell proliferation and death, migration, and differentiation that occurs during neural tube closure (NTC). In the proposed research program, we will test specific hypotheses using this mouse model: (1) Maternal supplementation with methionine, purines, thymidylate and S-adenosylmethionine can protect against NTDs in nullizygous Mthfd1l (Mthfd1lz/z) embryos, (2) Depakote (Valproic Acid; VPA), the leading cause of pharmaceutical-induced NTDs, inhibits mitochondrial 1C metabolism, thus it is possible that formate can prevent NTDs caused by this teratogen, and (3) cell proliferation and apoptosis, cell migration, and differentiation programs are disrupted in Mthfd1lz/z embryos, leading to neural tube and orofacial defects. We demonstrated that maternal supplementation of MTHFD1L dams with formate, the product of the MTHFD1L enzymatic reaction, decreases the incidence of NTDs and partially rescues the growth deficit in embryos lacking a functional Mthfd1l. In Specific Aim 1 we will determine which supplements downstream of the MTHFD1L reaction can rescue the NTD phenotype much like formate. This will be explored in a number of FA responsive and non-responsive NTD mutant strains, and in VPA-sensitive mouse strains. We will identify which cellular processes are dysregulated in Mthfd1lz/z embryos and VPA-sensitive mouse strains, leading to improper NTC. Metabolomics and epigenetic studies will be pursued to fully characterize the mutant mice.  Specific Aim 2 will focus on the requirement for formate in neural stem cells and neural crest stem cells using neurosphere growth and differentiation assays, as well as additional epigenetic investigations.  The final series of studies will involve DNA resequencing of the human MTHFD1L gene and performing functional analyses of identified variants in a spina bifida cohort. Our proposed research program offers hope for developing the first effective intervention for non-FA responsive NTDs by illuminating the underlying mechanisms by extremely important in preventing these preventable birth defects.


This work is supported by NIH grant R01HD083809 Intervention Strategies for Non-Folate Responsive Neural Tube Defects.  Funding Period: . 2/15/2016 – 1/31/2021
 

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Identification of Risk Genes and Environmental Interactions in the Etiology of NTDs

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This is a program project grant in collaboration with colleagues at Weill Cornell Medical College in New York.  The Finnell Laboratory is responsible for Project 3.
Neural tube defects (NTDs) arise from a complex interplay of multiple genes and environmental exposures. In human populations, folic acid (FA) supplementation can prevent up to 70% of NTD occurrences--including anencephaly and spina bifida—by as yet unknown mechanism(s). Nevertheless, FA fails to benefit at least a third of families and recent data suggest that in some specific genetic contexts, FA may be deleterious to the developing embryo. Clearly, families would be far better served if their individual risks could be accurately assessed, including identification of which aspect of the FA metabolic pathway--or which supplement involving another pathway entirely--would provide the most benefit to them, so that NTD prevention strategies could be optimized according to individual genetic risk factors.
This program aims to improve NTD risk assessment and prevention by integrating advanced human genomics with biological paradigms in humans and mice for identifying key gene-environment interactions. 


Project 1 (Ross PI with Finnell & Gross) has accumulated 200 whole genome sequences (WGS) from cases and 200 controls and has identified rare nonsense, frameshift and non-coding variants associated with spina bifida. In the renewal, we will employ a powerful high throughput method using molecular inversion probes (MIPs) to resequence a replication cohort of over 2,000 NTD cases. Cutting edge CRISPR-Cas9 dependent genome editing in hESCs and mice will probe the functional impact of identified variants on neuroepithelial cell polarity, proliferation, and the generation of reactive oxidative/nitrosative species (RONS).


Project 2 (Gross PI with Ross & Finnell) will test the hypothesis that a major role for folate protection against NTD is to suppress the generation of RONS. They will employ a novel untargeted stable isotope method to trace folate-mediated 1-C trafficking in NTD-susceptible mouse models. In addition, they will employ a novel redoxome platform to quantify oxidatively-modified small molecules in NTD prone mice. With Projects 1&3, they will examine the impact of identified NTD associated human variants on cellular redox status and 1-C trafficking and the extent to which supplementation with small molecules can modulate these actions.


Project 3 (Finnell PI with Gross & Ross) will examine the interaction of genetic variants and RONS to disrupt signaling pathways and cause cell damage during NT closure. They will test the ability of a human NTD-associated variant in NO synthase, NOS3, to increase ROS peroxynitrite in cells due to the phosphorylation of NOS3 on Ser633. It will test whether mitochondria are a major source of RONS during neurulation. Together, Projects 1, 2, & 3 will help define interactions of maternal/embryonic genetics, nutritional status and 1-C metabolism with NTD risk, using extensive human genomics, proteomics/metabolomics, and CRISPR-Cas9-dependent genome editing in hESCs, patient stem cells (iPSCs) and mice.

This work is supported by NIH grant P01HD067244. Risk Genes and Environmental Interactions in NTDs.  Funding Period: 7/1/2016 – 6/30/2021.
 

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How can we determine the forces that drive the neural folds to fuse and close the neural tube?

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The objective of this research program is to develop a non-contact, all-optical imaging technology to map elastic moduli and forces involved in critical aspects  of  embryonic  development  with  high  3D  resolution.  The proposed technology is based on combined Brillouin spectroscopy and Optical Coherence Tomography (OCT), which will be used to gain fundamental understanding of biomechanical factors involved during neural tube closure (NTC) in normal and pathological cases using murine neural tube defect (NTD) models.  As the neural tube gives rise to the adult brain and spinal cord, NTDs arise  when  a  failure  of  NTC  occurs.  NTC comprises a complex series  of  processes  that  involve motion, thus are driven by forces. However, the biophysics of NTC, namely the interplay between tissue forces and stiffness,  i.e.  the  local  resistance  of  tissue  to  being  deformed  by  an  applied  force,  remains  poorly understood, mostly because of sub-optimal measurement techniques.  In the past few years, in collaboration with the Kirill Larin research group at the University of Houston, we have utilized  advanced  imaging  technologies,  i.e.  OCT  that the Larin Lab developed for  structural/functional  imaging  of  developing  embryos and  Brillouin  microscopy  for  mechanical  mapping  of  tissues,  that,  when  combined,  can  be  transformative  to elucidate  the  biomechanics  underlying  the  development  of  NTDs.    Our long-term  goal is  to  elucidate  how mechanical  properties  controlling  NTC  in  developing  embryos  can  be  manipulated  to  ensure  proper  neural development  in  at  risk  embryos.  Our central  hypothesis is  that  failure  of  NTC  leading  to  neural  tube  defects (NTDs) is mediated by mechanical alterations and abnormal forces at the edge of the closing neural tube that can be imaged with Brillouin-OCT multimodality.  In this proposal, to test this central hypothesis, our objective is  to  combine  OCT,  Brillouin  microscopy  and  analytical  modeling  to  establish  a  platform  technology  to  map elastic  moduli  and  forces  in  developing  mouse  embryos.


  The  research  premise  of filling  the gap  of  our understanding  of  NTC  biomechanics  is  supported  by  our strong  preliminary  data.  To date we  have  shown  that  it is possible to assess the  dynamics  of  NTC  in  live  normal  and  mutant  embryos  using  OCT  and  map  the  heterogeneous  distribution of tissue stiffness in developing mouse embryos using Brillouin imaging (completely noninvasively with high 3D resolution). Our research efforts will initially focus on the advanced development of Brillouin microscopy to measure live  embryonic  tissue.  Combined  Brillouin/OCT  instrument will  be  developed,  and  we  will  test  the  hypothesis  that  mechanical  properties  and  forces  critically  mediate  genetically predisposed or teratogen induced NTDs. We hope that at the completion of this project, we will produce a unique platform technology that  will  enable  studies  where  a mechanical  phenotype  is  correlated  with  gene  and protein expression profiles in order to provide mechanistic understanding of the entire developmental spectrum of events leading to NTDs. 


This work is supported by NIH grant R01HD095520-01A1 Biomechanics of Neural Tube Development Using Brillouin-OCT Multimodality.  The funding period is 9/1/2018-8/31/2023.
 

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What are the roles of miRNAs in the Etiology of Neural Tube Defects?

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To date more than 300 genes have been identified that contribute to an NTD phenotype in mice, while the genetic basis of human NTDs remains unclear.  Commonly used testing to detect mutations in the setting of congenital malformations such as whole genome sequencing, do not reveal potential deleterious mutations in crucial regulatory elements, including microRNAs (miRNAs).  miRNAs are small, non-coding RNA molecules that have the ability to induce large-scale transcriptional changes through post-translational silencing of numerous RNA transcripts simultaneously.  Although they have been shown to regulate virtually every developmental and disease process, their role in neural tube formation has not been rigorously investigated.

The miR-302 family, the most highly expressed miRNA family in human and mouse embryonic stem cells (ESCs) is a well-established regulatory of pluripotency and cellular differentiation.  We recently found that mice lacking miR-302 have a fully penetrant NTD phenotype, demonstrating an essential role during neural tube closure, and showed that miR-302 regulates developmental timing and differentiation of neuroepithelial cells during neurulation.  In whole genome sequencing studies of human NTD cases, we discovered mutation in miR-302 , suggesting conserved function in mouse and human.  Expression profiling from miR-302 knockout and wildtype mice indicated that genes in the Wnt/planar cell polarity pathway and cilia/hedgehog pathways-two pathways that are critical for proper neural tube closure, are mis-regulated in miR-302 knockout neuroepithelium.  We hypothesize in this research program that miR-302 regulates neural tube closure and developmental timing of differentiation through the Wnt/PCP and ciliary/Hh pathways.


This work is supported by NIH grant R01HD098131-01A1 MicroRNA Regulation of Neural Tube Closure.  The funding period is: 03/01/2020-02/28/2025.
 

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Investigating the role of mitochondrial one carbon metabolism in the etiology of non-folate responsive NTDs.

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The etiology of NTDs are known to be multi-factorial, including genetic and environmental factors. There are multiple developmentally related signaling pathways involved as NTC proceeds spatially and temporally. The identification of specific genetic variants that contribute significantly to the etiology of NTDs, and the characterization of their underlying molecular and cellular mechanisms leading to failed NTC has progressed slowly over the last several decades. What is important to recognize is that NTDs stand out as a preventable birth defect. Research spanning decades demonstrates that maternal periconceptional supplementation with folic acid can reduce the risk of NTDs by 30% to 70%. Yet not all NTDs are folate responsive.  Currently between 30-50% of all NTDs are not preventable by folic acid supplementation. This project is based on our recently published folic acid non-responsive Slc25a32 null mouse model, and its interaction with a folic acid responsive Wnt co-receptor, Lrp6 mutant mouse model. The studies are designed to help elucidate the underlying mechanisms characteristic of folic acid resistant NTDs, and to test our hypothesis that these folate resistant NTDs may be prevented by interventions with downstream folate metabolites, such as formate. It was recently determined that formate could rescue folic acid resistant NTD mice suggesting that mitochondrial one carbon metabolism might be compromised in the non-folate responsive NTD population.   Despite almost 40 years of intensive study, we still do not fully understand the molecular, cellular and biochemical mechanisms that underlie the folate-dependent process of NTC. This gap in our knowledge hinders our ability to make informed health policy decisions about folate fortification and to identify novel treatments to prevent folate resistant NTDs.


This work is supported by NIH grant R01 HD100535-01 Role of Slc25a32 and Its Interaction with Lrp6 in the Etiology of Neural Tube Defects.  The funding period is: 03/01/2020-02/28/2025.
 

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Do Anti-Retroviral Therapeutic Agents Cause Neural Tube Defects?

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The human immunodeficiency virus (HIV) integrase inhibitors are increasingly being used for anti-retroviral therapy (ART), and dolutegravir (DTG) has emerged as a leading core agent. The DTG/Tivicay manufacturer reports (09/2018) that animal reproduction studies showed no evidence of adverse developmental outcomes, but an ongoing observational human cohort study in Botswana initially reported a 9-fold increase for neural tube defect (NTD) risk in offspring from mothers receiving DTG. With increased exposed births but no additional NTDs, a 6-fold increase for NTD risk in infants with early gestational exposure to DTG still remains. Recent concerns about teratogenicity have led to
caution for DTG-based regimen use in women of child-bearing potential. We hypothesized that if DTG is teratogenic, then gestational exposure to DTG will result in changes to one or more essential developmental processes, affecting functional mechanisms that have direct roles in neurulation and NTDs.

We report a mechanism of action (MOA) for DTG teratogenicity and demonstrate specificity of this MOA in an animal model by rescue of DTG-induced developmental toxicity. Competitive binding data indicates DTG is a partial antagonist of folate receptors at clinically relevant concentrations. Data
from the zebrafish model show developmental toxicity due to early embryonic exposure to DTG. Specificity of DTG developmental toxicity is demonstrated via rescue of DTG-induced developmental toxicity by supplemental folate. Folates and folate receptor are established modifiers of risk for NTDs,
and binding data indicates DTG is an inhibitor of folate receptor.

This work is supported by NIH grant R01 HD100229-01A1 Plausible Causative Mechanism for Dolutegravir Developmental Toxicity.  The funding period is: 11/01/2019-10/31/2024.
 

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CIC Gene Discovery

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Cerebral folate deficiency (CFD) syndrome is characterized by very low concentrations of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid, while folate levels in the plasma and red blood cells are within normal limits. Previously, mutations in several folate pathway genes, including hFR (folate receptor alpha), DHFR (dihydrofolate reductase), and PCFT (proton coupled folate transporter) have been identified in patients with low concentrations of 5-MTHF in their cerebrospinal fluid. In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis of DNA samples collected from a CFD patient, her healthy siblings, and her biological parents. A de novo mutation in the Capicua gene (CIC), c.1057C>T (p.R353X), was identified in the patient. The results were confirmed using Sanger sequencing. In addition, a missense mutation predicted to be damaging, c.1738G>GT (p.G580GC) was identified in another CFD patient. The CIC protein is a HMG-box transcriptional repressor. The DNA binding domain located at amino acid residues 200-268 binds the octomer sequence T(G/C)AATG(A/G)A. The mutation identified in the CFD patient, p.R353X, yields a truncated protein which still contains the DNA binding domain (HMG box); therefore, it is still able to bind to its targets. CIC target binding octomer sequence has been found in the promoter regions of folate transport genes FOLR1, PCFT, RFC1, and DHFR, which is involved in folate metabolism. In the patient’s induced pluripotent stem (iPS) cell, the p.R353X mutation down regulated FOLR1, PCFT and RFC1 gene expression compared with H9 stem cells and an iPS cell line from an individual with the wildtype CIC genotype. Chromatin immunoprecipitation assays demonstrated that CIC bound to the FOLR1, PCFT and RFC1 promoters in vitro. In a dual-luciferase assay, the CIC protein repressed FOLR1 promoter transcription. Using CRISPR-Cas9 technology, we have made a p.R353X knock –in mouse strain. It will be used to study whether this variant could cause CFD in mice, the underlying mechanisms and possible therapy strategies.