Effects of thyroxine exposure on the Twist 1 +/− phenotype: A test of gene–environment interaction modeling for craniosynostosis

Abstract

Craniosynostosis, the premature fusion of one or more of the cranial sutures, is estimated to occur in 1:1800-2500 births. Genetic murine models of craniosynostosis exist, but often imperfectly model human patients. Case, cohort, and surveillance studies have identified excess thyroid hormone as an agent that can either cause or exacerbate human cases of craniosynostosis. Here we investigate the influence of in utero and in vitro exogenous thyroid hormone exposure on a murine model of craniosynostosis, Twist 1 +/−. By 15 days post-natal there was evidence of coronal suture fusion in the Twist 1 +/− model, regardless of exposure. With the exception of craniofacial width, there were no significant effects of exposure; however, the Twist 1 +/− phenotype was significantly different from the wild type control. Twist 1 +/− cranial suture cells did not respond to thyroxine treatment as measured by proliferation, osteogenic differentiation, and gene expression of osteogenic markers. However, treatment of these cells did result in modulation of thyroid associated gene expression. Our findings suggest the phenotypic effects of the genetic mutation largely outweighed the effects of thyroxine exposure in the Twist 1 +/− model. These results highlight difficultly in experimentally modeling gene-environment interactions for craniosynostotic phenotypes.

Pharmacological exposures may precipitate craniosynostosis through targeted stem cell depletion

“…Similarities between human and murine craniofacial development make the wild-type model used here appropriate for this investigation. Without the complicating genetic predisposition to premature suture fusion, we are able to more specifically interrogate the effects of these pharmacological agents on suture maintenance (Durham et al, 2016). As it is possible that thyroid disorder and depression could spontaneously occur during pregnancy while nicotine use is more likely to occur before, after, and throughout gestation we have modeled clinically relevant conditions though our dosing schematics were different depending on the teratogen of interest.…”

“…Homeostasis of cells within the suture mesenchyme is vital to maintenance of the fibrous suture space between osteogenic fronts. Since nicotine is a known stimulant (Schaal and Chellappan, 2014; Thun et al, 2013; Cardinale et al, 2012), levothyroxine is known to preferentially affect cartilage cells (Howie et al, 2016; Durham et al, 2017b; Durham et al, 2016), citalopram is known to affect bone cells (Howie et al, 2018), understanding the cell activity with regard to osteogenic differentiation, programmed cell death, and proliferation is important to understanding how these pharmacological agents affect cells within the calvarial sutures. Immunohistochemical assessment of cell activity within the suture mesenchyme after levothyroxine exposure indicated a decrease in proliferation across both sutures, and a reduction in apoptotic activity compared to control in the coronal suture.…”

“…Primary, wild type suture cells were isolated as previously described (Durham et al, 2019; Durham et al, 2016), plated at a density of 65,000 cells per well in 6 well plates and treated for 7 days with control media (DMEM, supplemented with 10% FBS, 1% penstrep, and 0.2% amphotericin) or media supplemented with pharmacological agents (levothyroxine (780 ng/ml), nicotine (25 ng/ml), or citalopram (250 ng/ml)) at levels mimicking circulating levels for 7 days (Durham et al, 2019; Durham et al, 2015). Subsequently, total RNA was isolated using the Qiagen RNEasy mini kit (Qiagen, Valenica, CA, USA) according to manufacturer’s protocol.…”

Direct Effects of Nicotine Exposure on Murine Calvaria and Calvarial Cells

“…To determine the specific effect of nicotine on the cells comprising the calvarial growth sites and centers, primary, wild type coronal suture cells isolated as previously described 42 , murine bone marrow stem cells (BMSC) 43 , as well as isotype cell lines pre-osteoblasts MC3T3-E1 and chondrogenic cells ATDC5 (ATCC, USA), were cultured at 37 °C in a humidified 5% CO 2 incubator. MC3T3-E1 cells were cultured in αMEM (Lonza, USA), while the remaining cells were cultured in DMEM, supplemented with 10% FBS, 1% penstrep, and 0.2% amphotericin with media changes twice per week until 95% confluence was reached.…”

Gene/environment interactions in craniosynostosis: A brief review

“…Teratogenic studies alone are informative, but do not address interactive effects. Further, the addition of teratogens within a genetic model of craniosynostosis has several drawbacks including the strong possibility of no effect as craniosynostosis is already predicted to occur (i.e Twist 1 +/− mouse) . However, these studies in both the wild‐type mammalian skull and genetic models of craniosynostosis may provide sufficient information on molecular pathways of effect (downstream of the gene) specific to an exposure.…”