A tissue-specific role for intraflagellar transport genes during craniofacial development

RESEARCH ARTICLE A tissue-specific role for intraflagellar transport genes during craniofacial development Elizabeth N. Schock1,2☯, Jaime N. Struve1,2☯, Ching-Fang Chang1,2☯, Trevor J. Williams3, John Snedeker4, Aria C. Attia4, Rolf W. Stottmann2,4, Samantha A. Brugmann1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Division of Plastic Surgery, Department of Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 2 Division of Developmental Biology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 3 Department of Craniofacial Biology, University of Colorado School of Dental Medicine, Aurora, Colorado, United States of America, 4 Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Schock EN, Struve JN, Chang C-F, Williams TJ, Snedeker J, Attia AC, et al. (2017) A tissue-specific role for intraflagellar transport genes during craniofacial development. PLoS ONE 12(3): e0174206. https://doi.org/10.1371/journal. pone.0174206 Editor: Knut Stieger, Justus Liebig Universitat Giessen, GERMANY Received: December 8, 2016 Accepted: March 6, 2017 Published: March 27, 2017 Copyright: © 2017 Schock et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Primary cilia are nearly ubiquitous, cellular projections that function to transduce molecular signals during development. Loss of functional primary cilia has a particularly profound effect on the developing craniofacial complex, causing several anomalies including craniosynostosis, micrognathia, midfacial dysplasia, cleft lip/palate and oral/dental defects. Development of the craniofacial complex is an intricate process that requires interactions between several different tissues including neural crest cells, neuroectoderm and surface ectoderm. To understand the tissue-specific requirements for primary cilia during craniofacial development we conditionally deleted three separate intraflagellar transport genes, Kif3a, Ift88 and Ttc21b with three distinct drivers, Wnt1-Cre, Crect and AP2-Cre which drive recombination in neural crest, surface ectoderm alone, and neural crest, surface ectoderm and neuroectoderm, respectively. We found that tissue-specific conditional loss of ciliary genes with different functions produces profoundly different facial phenotypes. Furthermore, analysis of basic cellular behaviors in these mutants suggests that loss of primary cilia in a distinct tissue has unique effects on development of adjacent tissues. Together, these data suggest specific spatiotemporal roles for intraflagellar transport genes and the primary cilium during craniofacial development. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This research was supported by National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grant R01DE023804 (SAB), F31DE025537 (ENS), 1R01 DE019843 (TJW), and from R01GM112744 and March of Dimes Foundation 5-FY13-194 (RWS). Primary cilia are ubiquitous, microtubule-based extensions that protrude off a plethora of cell types throughout development. Interest in primary cilia biology has grown exponentially over the last decade, mostly due to the identification of ciliopathies, a growing class of human syndromes that occur as a result of aberrant cilia function [1]. Although there is no established phenotypic criterion for diagnosis of a ciliopathy, it has been hypothesized that a ciliopathy PLOS ONE | https://doi.org/10.1371/journal.pone.0174206 March 27, 2017 1 / 28 A tissue-specific role for intraflagellar transport genes during craniofacial development Competing interests: The authors of this manuscript have declared that no competing interests exist. could be defined on the basis of common phenotypic presentations. The initial suggestion for ciliopathic presentation consisted of a combination of nine common phenotypes including: retinitis pigmentosa, renal cystic disease, polydactyly, situs inversus, mental retardation, hypoplasia of the corpus callosum, Dandy-Walker malformation, posterior encephalocele and hepatic disease [1]. These common phenotypic characteristics suggest that certain tissues are particularly sensitive to the loss of primary cilia. More recently, the craniofacial complex has also been identified as an organ system particularly sensitive to the loss of primary cilia [2–11]. Approximately 30% of all ciliopathies are primarily defined by their craniofacial phenotype, which frequently includes cleft lip/palate, craniosynostosis, micrognathia/aglossia and midfacial hyperplasia [5]. The craniofacial complex is comprised of tissues from various embryonic origins including the neuroectoderm, neural crest and surface ectoderm. Defects in any one of these tissues can lead to severe craniofacial disorders, and null mutations would be expected to be most severe, as they would represent the combinatorial loss of function in all tissues. One of the barriers to gaining a clearer understanding of the etiology of ciliopathic phenotypes has been conflicting reports regarding phenotypes and molecular readouts of ciliopathic mutants. Specifically, there have been contradictory reports regarding how loss of cilia affects Hedgehog (Hh) signaling [12–16]. Some studies reported that loss of functional cilia produced a loss-of-Hh target gene expression [14, 16], while other data showed that loss of functional cilia produced a gain-of-Hh target gene expression [12, 13]. Conflicting data also exists when examining the role of individual ciliary proteins in Hh signaling [6, 15]. The basis for these conflicts are still under investigation; however, it is clear that the molecular mechanism rendering the cilia non-functional (e.g., which ciliary protein is lost or mutated) and the tissue in which this occurs has a major influence on the eventual effect on Hh target gene expression. This hypothesis is supported by the wide spectrum of phenotypes observed in ciliopathic animal models [5]. The axoneme of the cilium extends from the basal body and protrudes off the apical surface of the cell. Several ciliopathies are caused by the loss of axonemal extension, which requires intraflagellar transport (IFT) proteins. IFT proteins are divided into two classes: anterograde proteins (Class B) which carry molecular cargo from cell body to the ciliary tip (e.g., KIF3A and IFT88), and retrograde proteins (Class A) which carry molecular cargo from the ciliary tip back to the cell body (e.g., TTC21B). Our previous w (...truncated)