Fishing the Molecular Bases of Treacher Collins Syndrome

Citation: Weiner AMJ, Scampoli NL, Calcaterra NB ( Fishing the Molecular Bases of Treacher Collins Syndrome Andrea M. J. Weiner 0 Nadia L. Scampoli 0 Nora B. Calcaterra 0 Gary Stein, University of Massachusetts Medical School, United States of America 0 Instituto de Biolog a Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Cient ficas y Te cnicas (CONICET) - A rea Biolog a General, Departamento de Ciencias Biolo gicas, Facultad de Ciencias Bioqu micas y Farmace uticas, Universidad Nacional de Rosario , Rosario , Argentina Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development. - Funding: This work was supported by Agencia Nacional de Promocio n Cientfica y Tecnica (ANPCyT)-PICT 00648 and Universidad Nacional de Rosario (UNR)BIO19. AMJW is a fellow and NBC a staff member of CONICET and UNR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Treacher Collins syndrome, also known as mandibulofacial dysostosis (TCS, OMIM #154500), is an autosomal dominant craniofacial malformation affecting 1:10,000 newborns. The affected structures have a common embryological origin in the first and second branchial arches. The resulting phenotype is characterized by downslanting palpebral fissures, lower lid coloboma, malar and mandibular hypoplasia, high or cleft palate, external ear malformations, atresia of the hearing canal, and conductive hearing loss [1]. There is striking clinical variability, with the most severe cases leading to perinatal death due to respiratory distress, and mild cases that escape clinical diagnosis. Despite this large spectrum of variability, penetrance is thought to be complete [2]. Mutations in the Treacher Collins-Franceschetti syndrome 1 gene (TCOF1, OMIM *606847), mapped to chromosome 5q32-q33.1, are responsible for over 90% of TCS cases. Over 120 pathogenic mutations have been identified in TCOF1. This observation suggests that haploinsufficiency of TCOF1s protein product, treacle, is the underlying cause of TCS [3]. The majority of mutations in TCOF1 result in truncated treacle proteins [49], highlighting the importance of the C-terminal domain for treacle function. Treacle appears to participate in ribosome biogenesis by controlling pre-rRNA synthesis or processing [1012]; however, the treacle biological role has not been fully understood yet. A model of TCS was achieved by knocking-out the mouse Tcof gene. Homozygous are lethal and neonatal Tcof1+/2 mice die within 24 h of birth [13]. Tcof1+/2 mice show fewer mature ribosomes in neuroepithelial and neural crest (NC) cells [13,14], which were described as the expression territories of Tcof1 during embryonic development [13,15]. Recent results show that inhibition of p53 rescues craniofacial abnormalities by preventing apoptotic elimination of NC cells (NCC) [16]. Nevertheless, the ribosome biogenesis was not restored to wild-type levels in rescued mice [16]. This finding suggests that TCS phenotype is not only due to defects in ribosome biogenesis and, moreover, makes imperative to carry out further researches to fully-elucidate the etiology of this pathology. During the last two decades, the zebrafish has emerged as an important model for vertebrate development as it relates to human health and disease. Several woks using this animal system have provided significant insights into the variety of cellular mechanisms and tissue interactions necessary for proper craniofacial skeleton development [17]. Zebrafish forms essentially all of the same skeletal and muscle tissue types as its higher vertebrate counterparts, but in a simpler pattern, and tissues are composed of fewer cells [18]. In view of this, we have assessed the potentiality of zebrafish for studying the mechanisms responsible for the TCS pathogenesis. Here we report the identification of a zebrafish gene, formerly called B8JIY2 DANRE, as the TCOF1 ortholog. We cloned and analysed B8JIY2/tcof1 expression in developing zebrafish and detected a dynamic spatiotemporal pattern similar to that observed for Tcof1 in mouse. The knockdown of B8JIY2/ tcof1 expression adversely affected the NC development and, furthermore, resulted in fish showing typical features of TCS patients. Remarkably, only craniofacial development was affected since the rest of embryonic structures developed normally in B8JIY2/tcof1 knocked down zebrafish. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. Together, results strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 orthologous in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development. In silico identification of B8JIY2 as the putative ortholog of TCOF1 in zebrafish There was initially no information related to tcof1 gene in zebrafish genomic databases. Therefore, we began our analysis using the tcof1 cDNA sequence from Xenopus laevis (GenBank accession number AY731504) to search for homologous sequences in the Danio rerio cDNA_ALL Ensembl database (tblastx, Assembly Zv9). Two alignments with a high percentage of identity were located on a region of the minus strand of chromosome 13 (Table S1 and Figure 1A; Chromosome13: 4,655,6854,665,683). This region of chromosome 13 contains the Ensembl predicted genes B8JIY2_DANRE, Q7ZUM1_DANRE, an (...truncated)