Our Research

 Finnell/Cabrera Birth Defects Laboratory

Make Stacey Walk-Studies in the FKBP8 Knockout Mouse


One of the most ambitious programs ongoing in the laboratory involves utilizing cell free protein factors to help mitigate the co-morbidities associated with spina bifida in our FKBP8 knockout mouse model.  This program draws upon the diverse research strengths of the Finnell/Cabrera Birth Defects Laboratory, which includes: Dr. Jacqueline Parchem, a maternal-fetal medicine specialist who provides amniotic fluid, placental and umbilical cord tissues from which stem cells and exosomes are isolated for the experiments.  Dr. Bruna Corradetti, a nanotechnology expert prepares the exosomes and other cell free components for transplantation into the model system.  Dr. Bogdan Wlodarczyk and Dr. Ying (Linda) Lin perform the mouse surgeries to deliver the test compounds to the dam/embryos.  Drs. Richard Finnell and Robert Cabrera devise the testing strategies, examine the embryos, and perform related neurobehavioral assays. These experiments draw upon decades of preclinical and clinical research evaluating mesenchymal stromal/stem cell (MSC) utility which have demonstrated their capacity to promote endogenous tissue repair and regeneration in acute and chronic injury and disease. As spina bifida can be diagnosed before irreversible neurological damage has occurred, utilizing stem cells or cell free therapies have been proposed as a novel intervention for treatment/repair of spina bifida. The hope is for both for the application of regenerating neural tissue and protecting tissue from further damage. Moderately successful application of several repair strategies has been employed in rat models of spina bifida, using both direct microinjection into the spina bifida lesion or injection into the amnion, and using both rat and human MSCs. Additionally, human MSCs have been used to repair spina bifida in sheep models resulting in improved locomotor outcomes and motor function. As our studies with the FKBP8 knockout mouse progress, we have access to a herd of sheep with a high spontaneous prevalence of spina bifida available for transitioning to a large animal spina bifida model.


The Role of miR-302 in the Etiology of Neural Tube Defects


miRNAs are small, non-coding RNA molecules that have the ability to induce largescale transcriptional changes through post-transcriptional silencing of numerous mRNA 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 regulator of pluripotency and cellular differentiation. The Parchem Laboratory recently found that mice lacking miR-302 have a fully penetrant NTD phenotype, demonstrating an essential role during neural tube closure (NTC), 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 mutations in miR-302, suggesting conserved function in both the mouse and in humans. Expression profiling from miR-302 knockout (KO) and wildtype (WT) mice indicated that genes in the Wnt/planar cell polarity (PCP) and ciliary/hedgehog (Hh) pathways - two pathways that are critical for proper NTC - are mis regulated in miR-302 KO neuroepithelium. We hypothesize that miR-302 regulates NTC and developmental timing of differentiation through the Wnt/PCP and ciliary/Hh pathways.


In order to determine whether miR-302-associated human NTD variants have altered structure and function, we propose to examine miR-302 target regions that will be captured by PCR or molecular inversion probe enrichment and re-sequenced in genomic DNA samples from existing human NTD and control cohorts. This cohort has already yielded two NTD patients with miR-302 variants. Functional studies will be performed on the variants to determine the underlying pathogenic mechanism. Specifically, we will determine whether miR-302 variants disrupt microRNA secondary structure, regulation of target genes, cilia formation, neuroepithelial polarization, and neural tube formation using organoids. We hope that our  research program will establish miR-302 as a critical regulator of NTC leading to an improved understanding of the NTC process and the genetic links between Wnt/PCP, ciliary/Hh pathway and miR-302. This will promote the development of novel, targeted intervention strategies based on a more comprehensive understanding of the molecular mechanisms governing NTC and has broad implications for the 500,000 infants born with neural tube defects annually worldwide.


The Role of Planar Cell Polarity Genes in Complex Birth Defects


The planar cell polarity (PCP) genes have recently been associated with increased risks of neural tube and craniofacial malformations. In humans, polymorphisms in the PCP gene VANGL1 were found in several families affected by spina bifida and 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, including NTDs, suggesting an interaction with 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.

Using Embryonic Stem Cells to Screen for Reproductive Toxicants


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 chemical 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.

CIC Gene Discovery


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.