Disruption of the foxe1 gene in zebrafish reveals conserved functions in development of the craniofacial skeleton and the thyroid

Abstract

Introduction: Mutations in the FOXE1 gene are implicated in cleft palate and thyroid dysgenesis in humans.Methods: To investigate whether zebrafish could provide meaningful insights into the etiology of developmental defects in humans related to FOXE1, we generated a zebrafish mutant that has a disruption in the nuclear localization signal in the foxe1 gene, thereby restraining nuclear access of the transcription factor. We characterized skeletal development and thyroidogenesis in these mutants, focusing on embryonic and larval stages.Results: Mutant larvae showed aberrant skeletal phenotypes in the ceratohyal cartilage and had reduced whole body levels of Ca, Mg and P, indicating a critical role for foxe1 in early skeletal development. Markers of bone and cartilage (precursor) cells were differentially expressed in mutants in post-migratory cranial neural crest cells in the pharyngeal arch at 1 dpf, at induction of chondrogenesis at 3 dpf and at the start of endochondral bone formation at 6 dpf. Foxe1 protein was detected in differentiated thyroid follicles, suggesting a role for the transcription factor in thyroidogenesis, but thyroid follicle morphology or differentiation were unaffected in mutants.Discussion: Taken together, our findings highlight the conserved role of Foxe1 in skeletal development and thyroidogenesis, and show differential signaling of osteogenic and chondrogenic genes related to foxe1 mutation.

foxe1mutant zebrafish show indications of a hypothyroid phenotype and increased sensitivity to ethanol for craniofacial malformations

“…In our earlier study, we found FOXE1 in the thyroid follicles of developing zebrafish, which further bolstered the relation between FOXE1 and thyroid metabolism (Raterman et al 2023). In that study we found no differences in the number of thyroid follicles between mutants and wild type larval fish.…”

“…In mammals, hypothyroidism leads to delayed endochondral ossification, impaired growth, and reduced mineralization (Devdhar, Ousman, and Burman 2007; Bassett and Williams 2016; Persani 2012). Previously we reported that skeletal mineralization during endochondral ossification is decreased in foxe1 mutant larvae (Raterman et al 2023), but at that time we could not relate this to hypothyroidism yet. We now assessed a reduced mineralization and an increased osteoclast activity in scales from foxe1 and dio2 zebrafish mutants which we link to hypothyroidism.…”

“…Whole mount immunohistochemistry was performed on the scales exactly as described previously (Raterman et al 2023) using a Foxe1 antibody (Boster Bio (DZ41149), Pleasanton, USA) at 1:500. Z-stack images of the T4 staining were taken on an SPX8 confocal microscope (Leica).…”

“…Ethical approval for the experiments was granted by Radboud University's Institutional Animal Care and Use Committee (AVD10300202115245). Zebrafish foxe1^rdb2 mutants (Raterman et al 2023) and wild types were kept at standard care conditions (28°C under a 14 h light/10 h dark cycle) in the Radboud University Zebrafish Facility. Embryos and larvae were raised in petri dishes with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 , 0.00001% Methylene Blue) in an incubator at 28.5°C with a 14 h light/10 h dark cycle.…”

“…Recently, we successfully created a foxe1 mutant zebrafish that has a compromised Foxe1 function and altered expression of genes controlled by Foxe1 (Raterman et al 2023). Indeed, in these fish the symptoms of Bamforth-Lazarus syndrome are at least partly recapitulated; affected mutants show aberrant craniofacial development, most notably instability of the ceratohyal elements and decreased bone mineralization.…”