- Research article
- Open Access
RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome
© The Author(s). 2016
- Received: 2 November 2015
- Accepted: 26 May 2016
- Published: 13 June 2016
Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology.
Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development.
Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this mouse model. We have noted that a number of the dysregulated genes have known roles in brain development as well as epidermal differentiation and maintenance. Therefore, this study provides clues as to the underlying mechanisms that may be involved in the broader profile of WBS.
- Williams-Beuren syndrome
- Neurodevelopmental disorder
Gtf2ird1 is a member of the Gtf2i family of genes, encoding a set of multifunctional transcription factors. The three members of this family cluster within a domain of the 7q11.23 chromosomal region that is prone to copy number variation through non-allelic homologous recombination. Hemizygous deletion of this domain leads to the neurodevelopmental disorder, Williams-Beuren Syndrome (WBS) . This is a multisystem disorder with physical, cognitive and behavioural components. Studies of WBS patients with atypical deletions of the region have led to the conclusion that loss of GTF2IRD1 and GTF2I explain prominent features of the condition such as the craniofacial dysmorphology, the intellectual disability and the behavioural effects .
Analysis of mouse knockouts of the orthologous genes Gtf2ird1 and Gtf2i support these conclusions. Homozygous loss of Gtf2i causes embryonic lethality  but heterozygous loss results in increased anxiety, as measured by the light–dark box and elevated plus maze tests . Homozygous Gtf2ird1 mutants show reduced levels of aggression in the resident intruder test  impaired motor coordination and exploratory behaviour [6, 7] and altered vocalization in response to specific stress-inducing cues . Homozygous loss of Gtf2ird1 has also been shown to cause craniofacial abnormalities that in a transgene insertion mutant, affects the alignment of the jaws , but in the knockout model presented in this work, is mainly confined to an excessive overgrowth of the soft tissue . This phenotype is not apparent in the heterozygous Gtf2ird1 knockout mice. Detailed analysis of adult homozygous null Gtf2ird1 tm1Hrd mice demonstrated that these abnormalities were confined to the regions around the lips and the nose and histological sections showed that the increased thickness was due to an enlargement of the epidermal layer that sometimes resulted in skin flaps and folds in the lips and around the nares . While no histology of the face is available from WBS patients, it has been noted that the majority of the WBS craniofacial phenotype is soft tissue related .
Gtf2ird1 expression in the mouse is consistent with a role in craniofacial development and brain function . The Gtf2ird1 transcript is detectable from early stages of embryogenesis through to the development of specific tissues including cartilage, muscle, heart, brain and tooth buds. In the developing head, many of the hard and soft tissue components express Gtf2ird1. In the transition to maturity, many of these sites are shut down and expression in the adult becomes mainly confined to sensory organs, neurons of the peripheral and central nervous system, smooth muscle, cardiac muscle, cells in the testis and brown adipose tissue [9, 10].
Several studies illustrate GTF2IRD1’s DNA binding properties [11, 12] and its ability to act as a transcriptional regulator in transgenic mouse systems , as well as a capacity to auto-regulate its own transcription through direct binding of the GTF2IRD1 protein to its own promoter region . However, much of the information regarding GTF2IRD1 gene targets remains unknown. Analyses of Gtf2ird1 knockout brain tissue, for example, have led to a disappointing lack of useful information . Other studies have examined gene expression differences in mouse embryonic fibroblasts (MEFs) that overexpress Gtf2ird1 [16, 17] and in a Gtf2ird1 gene-trap mutant mouse model  that shows phenotypic defects that are more extreme than the Gtf2ird1 deletion models [5, 6, 8, 14]. This is the first published report of a comprehensive RNA-Seq analysis of the transcriptome of mice deficient for Gtf2ird1.
In this study we have analysed lip tissue from a Gtf2ird1 knockout mouse model in order to capture effects that are most apparent in the epidermis. Our observation that certain dysregulated genes have roles both in skin and brain development suggests that this analysis can provide insight into molecular effectors and pathways involved in WBS.
RNA-Seq analysis shows widespread gene dysregulation in the Gtf2ird1 KO
RNA was extracted from mouse lip tissue of three female Gtf2ird1 knockout mice (KO) and three female wild type mice (WT). The KO mice had a distinct physical appearance with overgrowth and wrinkling of the lip tissue, consistent with the phenotype previously described . The six RNA-Seq libraries were sequenced on the Illumina HiSeq2000 platform to produce over 60 million, 100 nucleotide paired-end reads per sample. Differential expression analysis was performed using the Bioconductor packages, edgeR  and subsequently confirmed using DESeq2 , as detailed in Methods. In this paper, we describe the RNA-Seq results broadly and identify differentially expressed genes with roles in normal epidermal and neurological development.
Differential gene expression seen in both directions in the Gtf2ird1 KO
Top 50 up-regulated and down-regulated genes found using edgeR
The genes listed in Table 1 have functions in transcription regulation (Foxq1, Sebox, Csrp3, Vgll2, Zbtb16, Sp110), signalling (Wnt7, Chrm3, P2ry4, Arhgap8, Au021092, Lypd6, F3, Skint10, Tac4, Gfra2, Dcc, Lrrn4, Defb13, Tie1, Ush2a), calcium binding (Pdlim3, Acsm1, Alox15, Cacng1, Capn9, Csrp3, Cyp2f2, Gm1110, Zbtb16), membrane transport (Slc6a19, Slc46a1, Aqp9), apoptosis (Csrp3, Zbtb16, Sp110, Dcc, Actc1), neurological processes (Chrm3, Dcc, Gfra2, Slc6a19, Ush2a) and development and maintenance of the stratified epidermis (Aqp9, Krt2).
Several transcription factors identified in the differentially expressed genes
Differentially expressed transcription factors
Downstream targets of Lhx2 found to be differentially regulated
Functional analysis of all the differentially expressed genes using gene ontology analysis and gene set enrichment
GSEA analysis of biological process and Kegg pathways most highly ranked gene sets
GSEA Biological process gene set (Up-regulated genes)
GSEA Biological process gene set (Down-regulated genes)
Anatomical Structure Development
Cell Cycle Process
Cell Cycle Go0007049
Cell Cycle Phase
Multicellular Organismal Development
Mitotic Cell Cycle
Cell Cell Signaling
M Phase Of Mitotic Cel Cycle
Rna Metabolic Process
Mrna Metabolic Process
Anatomical Structure Morphogenesis
Mrna Processing Go0006397
Striated Muscle Contraction Go 0006941
Response To Dna Damage Stimulus
Transmission Of Nerve Impulse
Chromosome Organization And Biogenesis
Amine Metabolic Process
Ribonucleoprotein Complex Biogenesis And Assembly
Nervous System Development
Neurological System Process
Generation Of Precursor Metabolites And Energy
Regulation Of Growth
Regulation Of Cell Growth
Response To Endogenous Stimulus
Nitrogen Compound Metabolic Process
Establishment Of Cellular Localization
Metal Ion Transport
Cell Cycle Checkpoint Go 0000075
Calcium Independent Cell Cell Adhesion
Transforming Growth Factor Beta Receptor Signaling Pathway
GSEA Kegg Pathways gene set
GSEA Kegg Pathways gene set
Kegg Parkinsons Disease
Kegg Oxidative Phosphorylation
Kegg Cytosolic Dna Sensing Pathway
Kegg Alzheimers Disease
Kegg Nod Like Receptor Signaling Pathway
Kegg Ecm Receptor Interaction
Kegg Nucleotide Excision Repair
Kegg Basal Cell Carcinoma
Kegg Rig I Like Receptor Signaling Pathway
Kegg Huntingtons Disease
Kegg T Cell Receptor Signaling Pathway
Kegg Cardiac Muscle Contraction
Kegg Cell Cycle
Kegg Hedgehog Signaling Pathway
Kegg Rna Degradation
Kegg Neuroactive Ligand Receptor Interaction
Kegg Toll Like Receptor Signaling Pathway
Kegg Dilated Cardiomyopathy
Kegg Aminoacyl Trna Biosynthesis
Kegg Glycosaminoglycan Biosynthesis Keratan Sulfate
Kegg Antigen Processing And Presentation
Kegg Hypertrophic Cardiomyopathy Hcm
Kegg Ubiquitin Mediated Proteolysis
Kegg Focal Adhesion
Kegg Primary Immunodeficiency
Kegg Calcium Signaling Pathway
Kegg Dna Replication
Kegg Arrhythmogenic Right Ventricular Cardiomyopathy Arvc
Kegg Non Homologous End Joining
Kegg Ppar Signaling Pathway
Kegg Homologous Recombination
Kegg Glycosphingolipid Biosynthesis Ganglio Series
Kegg Basal Transcription Factors
Kegg Pyrimidine Metabolism
Kegg Glycosphingolipid Biosynthesis Lacto And Neolacto Series
Kegg Epithelial Cell Signaling In Helicobacter Pylori Infection
Kegg Glycosaminoglycan Biosynthesis Chondroitin Sulfate
Kegg Vascular Smooth Muscle Contraction
Kegg Mismatch Repair
Kegg Other Glycan Degradation
Kegg One Carbon Pool By Folate
Kegg Wnt Signaling Pathway
Signalling pathways are dysregulated in Gtf2ird1 knockout tissue
We see down-regulation of members of the transforming growth factor family, Tgfb2 and the bone morphogenetic proteins, Bmp4 and Bmp6. Stimulation of the canonical pathway via TGFβ or the BMPs typically results in phosphorylation and activation of the SMAD transcription factors . We find no significant change in expression of the activating SMADs. Down-regulation of Tgfb2, which is an inhibitor of cellular proliferation, is consistent with the other evidence of increased cell division and proliferation seen in the KO phenotype .
The Wnt genes, Wnt7b, Wnt10b, and Wnt11 are all down-regulated in the KO. The most significantly down-regulated of these is Wnt7b, which is active in the canonical Wnt pathway. This is followed by Wnt11, which activates the non-canonical Wnt-calcium pathway that affects cytoskeletal dynamics and cell adhesion [24, 25] and Wnt10b, known to promote the differentiation of skin epithelial cells and the development of hair follicles . More generally, we find that a number of genes involved in calcium signalling are differentially expressed in the KO, as listed in Additional file 4: Table S4.
Increased growth factor signalling might be expected to result in increased cellular proliferation. We note that several of the up-regulated genes are involved in creating the cytoarchitecture of microtubules required for cell division during the mitotic phase of the cell cycle. For example, we see up-regulation of genes involved in cytokinesis or spindle formation and stabilization, such as Prc1, Anln, Aspm, Cenpe, Spdl1, Kif11, and Spag5. We also see increased expression of genes involved in chromatin condensation and the correct segregation of chromosomes during cell division, such as Sgol1, Smc1a, Smc2, Smc3 and Smc4. (Additional file 4, Table S5).
Genes known to be regulated by GTF2IRD1 and other known interaction partners of GTF2IRD1
The large number of differentially expressed genes identified in this study contrasts with the small number of direct interaction partners of GTF2IRD1 that are currently annotated, in the MetaCore and BioGRID databases (http://thebiogrid.org). The targets listed in the MetaCore database are primarily genes thought to be transcriptionally regulated by GTF2IRD1. The interactions listed in the BioGRID database are protein targets identified by Affinity-Capture and Two-hybrid methods. Our search of the MetaCore database produced 16 interaction partners and our search of the BioGRID databases produced 13 interaction partners. Our search results can be found in Additional file 5: Table S5. We recently reported work involving an unbiased screening strategy for interaction partners of GTF2IRD1 to rectify this shortcoming . Our study identified 38 novel interaction partners that are mostly involved in chromatin modification and transcriptional regulation as well as proteins associated with the primary cilium .
We have looked at whether any of the identified GTF2IRD1 interaction partners are differentially expressed in this RNA-Seq study, since the genes that encode these proteins may belong to a regulatory gene network that is active at the transcriptional and post-translational levels. Among the genes identified in the MetaCore™ and BioGRID databases we find decreased expression of two genes that have been implicated in transcriptional activation by GTF2IRD1, Ccnd3 (Cyclin D3) expression is decreased by 30 % (FDR = 0.065) and Tgfb2 (Transforming growth factor beta 2) decreased by 45 % (FDR = 0.002). Ccnd3 plays an important role in cell cycle progression and in phosphorylation of RB1. In addition to its interaction with RB1 and GTF2IRD1, CCND3 also affects other transcription factors and may have a repressive effect (for example on Runx1  and the androgen receptor ) or may have an activating effect such as for Pparg , Rara  and Vdr . TGFβ2 plays a role in the process that leads to the phosphorylation of SMAD, its translocation to the nucleus and ultimately transcriptional activation .
Our RNA-Seq results show a significant increase in expression in the Gtf2ird1 KO of Kpna2, Atf7ip, Parpbp which code for proteins identified by Carmona-Mora et al. (2015). We note that Trip11 and Alms1 are also increased but at an FDR of 0.061 and 0.065 respectively. It is interesting that each of these genes has been reported as having an association with the cell cycle. Atf7ip encodes a transcriptional coactivator or corepressor involved in cell cycle arrest [34, 35]. Parpbp plays a role in chromatin modulation, DNA repair and cell cycle progression . KPNA2 participates in nucleocytoplasmic transport and is associated with the cell cycle and DNA repair . ALMS1 has a role in the cell cycle through its involvement in formation and maintenance of cilia. TRIP11 is involved in the microtubule network and in transporting proteins to the ciliary membrane [38, 39].
Validation of RNA-Seq results with RTqPCR
All of these genes tested, with the exception of Aqp9 which had high variation in the KO samples, were found to be significantly differentially expressed in conformity with our RNA-Seq results.
In this work, we analysed the transcriptome of lip tissue from a Gtf2ird1 knockout mouse line. This line was created to better understand the effect of loss of the human orthologue of this gene in WBS, which is a neurodevelopmental disorder caused by the hemizygous deletion of GTF2IRD1 and other genes within the Williams-Beuren syndrome critical region of 7q11.23. Prior studies point to GTF2IRD1 affecting craniofacial development, cognition and behaviour [3, 5, 6, 8].
Using RNA-Seq, we have identified a large number of up-regulated and down-regulated genes, revealing that the inactivation of Gtf2ird1 has widespread effects. Our functional analysis indicates that these changes in gene expression may impact structural/morphological development of tissue, cellular differentiation, cellular proliferation and the immune response and that these processes may be mediated through aberrant signalling. Key molecular factors in development are transcription factors, signalling molecules and cell adhesion molecules and in this study we detect gene dysregulation in all three categories. We note overlap between the gene ontology terms highlighted in this study and those emerging from earlier microarray studies, indicating involvement of signalling, cell cycle and immune response in WBS gene dysregulation [17, 41].
Although GTF2IRD1 is known to regulate gene transcription [16, 42, 43], a relatively small number of direct targets of GTF2IRD1 have been identified to date. GTF2IRD1 has been shown to complex with the transcriptional repressor RB1, leading to the suggestion that it may be a general transcription factor, regulated through its association with RB1 and involved in cell-cycle progression . RB1 controls cell cycle progression by interacting with the E2F family, preventing the cell from progressing from G1 to S phase and by attracting histone deacetylases (HDACs) to chromatin, thus suppressing DNA synthesis. It has also been demonstrated that GTF2IRD1 associates with HDAC3 and to a lesser extent with HDAC1 . It has been suggested that the transcriptional activity of the GTF2I family may be modulated by the HDAC proteins during development, with this regulation being further refined by the activity of PIAS2 (an E3-type small ubiquitin-like modifier [SUMO] ligase) which associates with HDAC3 . Interestingly, we do see a strong signature of dysregulation in the cell cycle with up-regulation of many genes involved in cell division and proliferation. This observation, in the absence of any change in Rb1 expression, could be explained by a change in the post-translational modification of RB1. In this study, we see a decrease in Ccnd3 (FDR = 0.065) a gene that encodes a protein involved in the phosphorylation of RB1. An alternative or ancilliary explanation is that GTF2IRD1 may normally assist RB1 to find or bind to its targets and that its absence results in dysregulation of genes downstream of RB1, observed as increased cellular proliferation.
If GTF2IRD1 plays a direct role in cell proliferation through these mechanisms, one must ask why excess proliferation has not been seen in other tissues of the knockout mice where Gtf2ird1 is normally expressed. One possible explanation is that, unlike skin, these other sites undergo terminal differentiation and are not part of a system that undergoes continuous renewal through the activity of stem/progenitor cells and are therefore not in a position to respond to the change in GTF2IRD1 status in this way.
GTF2IRD1 has previously been associated with transcriptional repression [13, 14] which predicts that the majority of direct targets would show an increase in expression in the KO. It is noteworthy that the most statistically significant differentially expressed genes identified in this study are within the up-regulated group.
Our analysis points to dysregulation of a number of transcription factors involved in tissue development. We have drawn attention to one of these factors Lhx2, which may be especially relevant to WBS as it is active in tissue development and in neurological processes. LHX2 regulates hair follicle development  and skin repair and has been described as a central link in the genetic networks that coordinate multiple signalling pathways controlling organ development and cell fate determination . LHX2 also regulates brain development  and is an activator of SOBP  which has been observed to have strikingly specific expression in the limbic system, with disruption leading to abnormal cognition and intellectual disability . The putative effects of LHX2 seem highly relevant to WBS. We have therefore validated change in expression of Lhx2 with qPCR. In addition, our RNA-Seq analysis showed corresponding expression changes in many genes normally regulated by LHX2.
The dysregulation of genes that encode signalling molecules, which ultimately impact on transcriptional regulation, would also be predicted to contribute to the large number of differentially expressed genes detected in this study. This includes factors like CCND3, which has a repressive effect on Runx1  and the androgen receptor  and an activating effect on Pparg , Rara , Vdr  and TGFβ2 through its actions in the SMAD pathway.
Histological analysis has revealed dysfunctions in cellular proliferation and differentiation in the skin overlying the lips in Gtf2ird1 knockout mice . It is possible that these dysfunctions stem from dysregulation of signalling pathways. We have found altered expression of genes involved in multiple signalling pathways including growth factor signalling, Wnt, BMP and hedgehog signalling. These pathways act in a highly coordinated manner during the development of many tissues, although the mechanisms of cross-talk are still areas of active research [49–51].
Several members of the fibroblast growth factor (FGF) family are dysregulated (Additional file 4: Table S1). The FGFs regulate development by orchestrating mesoderm patterning in the early embryo and then guiding organ development by regulation of cell proliferation, differentiation and survival . Genes from this family are expressed in keratinocytes throughout all layers of the epidermis where they stimulate skin activity including induction of the induction of keratinocyte proliferation .
Members of the FGF family instigate signalling though the canonical mitogen-activated protein (MAP) kinase pathway, the STAT pathway, the PI3 kinase/AKT pathway and the PLCγ pathway . The MAP kinase pathway is involved in cell growth and differentiation, the PI3K pathway is implicated in cell survival and polarity control and the PLCγ pathway may be necessary for cell adhesion . Therefore, the fibroblast growth factors have a wide impact on biological activities that are of direct relevance to the epidermal phenotype seen in the KO [52, 54].
Fibroblast growth factor signalling also plays a critical role in brain development. Gene inactivation studies have shown that the receptors FGFR1 and FGFR2 are necessary for brain development  and proper formation of the medial prefrontal cortex and its connections with the limbic circuits . As well as seeing altered expression in Fgf1 and Fgfr1, we also see a 46 % decrease in Fgfbp3, a gene that has been associated with the regulation of anxiety . It is plausible that some of the gene expression changes seen in this study also occur when GTF2IRD1 levels are reduced in the brain, leading to dysregulation of FGF signalling and its functions in limbic system control. This could explain some of the behavioural abnormalities that are characteristic of WBS.
The interplay between FGF signalling and other signalling pathways is complex and will differ from tissue to tissue. However, it is known that the signalling pathways stimulated by the FGFs may also be stimulated by TGFβ, BMPs (the bone morphogenetic proteins) and Wnt ligands . In addition to changes in FGF signalling genes, we report decreased expression of Tgfb2, BMPs and Wnt signalling genes in the KO.
Overall, it appears that signalling through these ligands is decreased in the absence of Gtf2ird1. We therefore suggest that biological outcomes such as TGFβ-induced epithelial-to-mesenchymal (EMT) transition and TGFβ-induced axonal outgrowth may be affected. The TGFβ proteins generally have an anti-proliferative effect in epithelial cells as well as regulating the immune response. Amelioration of this anti-proliferative action is consistent with the hyperproliferation of epidermal cells seen in the Gtf2ird1 KO phenotype .
The Wnt signalling pathway is also involved in the same key biological processes of cellular proliferation, differentiation, adhesion and survival in a range of tissues, and plays an important role in the nervous system . The Wnt proteins mediate transduction in three major pathways, the canonical Wnt/β-catenin pathway, the planar cell polarity pathway and the Wnt-calcium pathway . In each of these pathways, the Wnt ligand binds to its cognate receptor, Frizzled. In this study, we find that genes encoding four of the Frizzled receptors (Fzd1, Fzd2, Fzd5 and Fzd9) are significantly down-regulated in the KO. The most significantly down-regulated Wnt gene is Wnt-7b, which is active in the canonical Wnt pathway. This is followed by Wnt-11, which activates the non-canonical Wnt-calcium pathway that affects cytoskeletal dynamics and cell adhesion [24, 25], and Wnt-10b, known to promote the differentiation of skin epithelial cells and development of hair follicles . We find decreased expression of genes in the Wnt-calcium pathway, as well as in calcium ion homeostasis. This is interesting as calcium signalling pathways are known to be key in keratinocyte proliferation, migration and differentiation , inflammation  and the nervous system , all relevant to the WBS phenotype.
Patients with WBS display a number of cognitive and behavioural abnormalities. In this study, we have observed that the differentially expressed genes are enriched with gene ontology terms relevant to neural processes. FGF signalling, Wnt signalling, and other forms of calcium signalling are key in a number of neural processes including neurogenesis, axon outgrowth and guidance, neuronal polarity, dendrite development, dendritic spine differentiation and synapse formation and maintenance . It is interesting that the calcium regulated oxytocin receptor is down-regulated in the KO. Dysregulation of oxytocin signalling is known to affect human behaviours involving trust and bonding , which may be particularly relevant to WBS .
It must be borne in mind that we have only tested lip tissue in this study. However, it is plausible that the mechanisms of GTF2IRD1 regulation that are disrupted here also operate in other tissue types including the brain. Microarray analysis of Gtf2ird1 knockout brain tissue has so far failed to identify significant levels of transcriptional dysregulation . However, the brain is a particularly difficult system for such investigations due to the complexity and cellular diversity; any alterations that occur within neuronal sub-populations are very hard to isolate and observe. It is useful, therefore, to generate hypotheses in proxy tissue systems, such as the lip tissue used here, and then apply these hypotheses in more specific ways to the brain.
In summary, we see widespread gene dysregulation in the lip tissue of mice in which Gtf2ird1 has been inactivated. We suggest that the loss of GTF2IRD1 has a direct effect on the expression of some genes, as well as altering the transcriptional control of other genes through the disruption of downstream transcription factors and signalling pathways. The combined effect leads to widespread gene dysregulation affecting multiple developmental mechanisms.
We have demonstrated that RNA-Seq is a powerful method for investigating global transciptomic changes resulting from Gtf2ird1 inactivation. This analysis directs our attention to mechanisms at play in neurodevelopmental conditions such as WBS, which involves the hemizygous deletion of GTF2IRD1 as well as other genes. Overall, we see widespread changes in expression of genes involved in tissue development and functional maintenance. This is evident in the samples of epidermal tissue analysed in this experiment but also has implications in other tissue such as the brain. Several of the genes found to be dysregulated play fundamental roles in a range of tissues including the brain and may be highly relevant to WBS. While the genes found to be dysregulated help to elucidate the processes involved in WBS, we are some way from understanding the mechanism by which GTF2IRD1 brings about these changes in gene expression. It is possible that GTF2IRD1 engages in multiple interactions with other nuclear factors and the complexes that are formed become located at a variety of genomic loci in order to regulate transcription. If the primary targets of GTF2IRD1 are other transcription factors and genes involved in signalling pathways, this could account for the broad gene dysregulation seen in this study. A clearer understanding of GTF2IRD1 function should, therefore, emerge from studies aimed at identifying protein-protein interactions, transcriptomics in a variety of tissues and assays designed to identify direct gene targets.
Gtf2ird1 tm1Hrd mice, referred to as knockout (KO) mice, were described previously . The mutation has been maintained on a C57BL/6 J background for greater than 20 generations and the experiments involved use of mice on this background. All experimental procedures were approved by the Animal Care and Ethics Committee at UNSW Australia. The mice used in the RNA-Seq and qPCR experiments were all adult females (2–6 months).
After mouse euthanasia by cervical dislocation, lip tissue from KO and WT mice were carefully cleaned with 75 % ethanol and DEPC-treated water. The top lip was then dissected and immediately immersed in 2 mL of cold TRI-reagent (Sigma). Lip tissues were homogenised until fully dissociated (approximately 30 s) using a T10 Ultra-Turrax homogeniser (Themo-Fisher Scientific) for subsequent RNA extraction. Tail biopsies were collected to confirm that the genotyping results established at 3 weeks old were correct.
Total RNA extraction
RNA was extracted from the dissected mouse tissues using TRI-reagent (Sigma), following the manufacturer’s instructions, which included a chloroform phase separation and ethanol precipitation. RNA was resuspended in 70 μL of RNase-free water. RNA samples were assessed for quantity and quality using a NanoDrop UV spectrophotometer (Thermo Fisher Scientific Inc), conventional RNA electrophoresis and using a Bioanalyser (Agilent Technology Inc). All RNA integrity numbers (RIN) for the analysed samples ranged from 9 to 9.5
RNA extracted from 3 WT mice and 3 KO mice was used to prepare six mRNA libraries following the standard Illumina protocol. The six RNA-seq libraries were sequenced on the Illumina HiSeq2000 platform at the Ramaciotti Centre for Genomics UNSW, to produce over 60 million, 100 nucleotide paired-end reads per sample (Reads 1 and 2).
Mapping RNA-Seq reads
The reads were mapped to the Ensembl Mus musculus genome (GRCm38) provided by Illumina iGenomes (downloaded from cufflinks.cbcb.umd.edu/igenomes.html). Mapping was performed with Tophat2 (v 2.0.8)  calling Bowtie2 (v 2.1.0)  using the default settings.
HTSeq-count (Python package HTSeq, python v 2.7.3) was used to generate counts of reads uniquely mapped to annotated genes using the GRCm38 annotation gtf file. HTSeq-count produced over 20 M uniquely aligned reads per sample (WT1 = 27811674, WT2 = 22050557, WT3 = 23415253, KO1 = 20164081, KO2 = 29491767, KO3 = 22204515). 26814 genomic features were counted with at least one read.
Differential gene expression analysis
We performed differential expression analysis using the count based method, edgeR (v 3.8.6)  and we confirmed these results using DESeq2 (v 1.6.3) , both tools are available as Bioconductor packages. Tables of raw counts generated using HTSeq-count (described above) were used as input in both analyses.
In the edgeR analysis, low count transcripts were excluded and only those genes with at least 1 count per million (cpm) in at least 3 samples were used for analysis. A normalization factor was calculated using the trimmed mean of M values (TMM) method  and the dispersion parameter for each gene was estimated using the quantile-adjusted conditional maximum likelihood (qCML) method, appropriated for experiments with a single factor. The functions estimateCommonDisp() and estimateTagwiseDisp() were used to estimate dispersion. Following this we tested for differential expression using the exact test based on qCML methods. The Benjamini-Hochberg correction was used with a false discovery cut-off of 0.1.
DESeq2 uses a generalised linear model (GLM) to assess differential expression. Dispersions were estimated using a Cox-Reid adjusted profile likelihood and the Wald test for significance of GLM was used. Automatic filtering is incorporated to exclude low abundance genes in the testing process.
We used the BiNGO plug-in to Cytoscape  to investigate the functional associations of genes found to be either down-regulated or up-regulated. As background we used the set of 14,526 genes used in our differential expression analysis. Of these, 13,376 genes were used in the gene ontology analysis. The Gene Ontology (GO) terms in the category Biological Process were tested for overrepresentation using the hypergeometric test and p values were corrected using the Benjamini & Hochberg FDR correction. Enriched GO were selected using a corrected p value of 0.01.
We used the REVIGO tool  to visualize the gene ontology terms associated with the differentially expressed genes.
We used the GseaPrePranked tool to indentify the enriched gene sets in c5.bp.v5.1.symbols.gmt of MSigDB (biological process) and in c2.cp.kegg.v5.1.symbols.gmt of MSigDB (Kegg pathways) with a permutation of 1000. An FDR q-value were of 0.05 was adopted .
We used MetaCore™ from Thomson Reuters to interrogate the list of differentially expressed genes for associated transcription factor factors. This allowed us to identify 78 transcription factors which are differentially expressed. MetaCore™ comprises a suite of software and an extensive database, which contains manually curated information on proteins, genes, complexes, metabolites, RNA and DNA and their interactions gleaned from the published literature. We also used MetaCore™ to find interaction partners of genes of interest.
We used BiNGO to obtain gene ontology terms associated with the differentially expressed transcription factors.
Validation with RTqPCR
First-strand cDNA synthesis was carried out using the SuperScript® III Reverse Transcriptase (Invitrogen™), with Random Hexamer Primer (Thermo Scientific™) using 1 μg of total RNA as template, according to the manufacturer’s instructions.
Quantitative real time-PCR
CDNA was prepared from four lip tissue RNA samples per genotype (4 adult WT mice and 4 adult KO mice).
Small samples of the cDNA (1–2 % of the total) were used as a template for quantitative PCR (qPCR) using the Fast SYBR® Green Master Mix (Applied Biosystems™). Reactions were set up to a total volume of 10 μL according to the product protocol and performed on the Stratagene MX3005P qPCR system (Agilent Technologies). Each reaction was set up in triplicate for the target gene under test. Triplicate reactions were also set up with an identical amount of template using primers designed against mouse Hprt (hypoxanthine phosphoribosyltransferase 1) as a housekeeping gene reference standard (see Additional file 6 for primer sequences).
For all RTqPCR assays, the efficiency of the different primer sets was tested by establishing a standard curve using serial dilutions of a cDNA pool made by combining samples of all the templates used in each experiment. MxPro QPCR Software was used to analyse the dissociation and amplification curves of every experiment and to obtain the threshold cycle values (Ct). Data were then analysed using Microsoft Excel for quantitation of target gene relative to the reference standard using the 2- ΔΔCT method . T-test analyses were performed on the 2- ΔΔCT values for the two groups of samples.
This project was assisted by a Project Grant from the National Health and Medical Research Council of Australia in favour of SP (1049639). CC and PC were funded by CONICYT Becas-Chile PhD scholarships. SC and MW were supported through funding allocated under the Australian Federal Government CRIS scheme, the NSW State Government RAAP scheme and UNSW.
Availability of data and materials
The raw data on which this publication is based are available at the Gene Expression Omnibus with the reference GSE81082.
The study was conceived by SP. SP and EH supervised the design and coordination of the study. The molecular biology, sample preparation and RTqPCR was carried out by CC and PC under the supervision of SP and EH and by LR under the supervison of AB. SC designed and performed the bioinformatic analysis under the supervision of MW. SC drafted the manuscript. SP, MW, CC, PC, LR and AB revised the draft and made contributions to the final manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics Approval and consent to participate
All experimental procedures were approved by the Animal Care and Ethics Committee at UNSW Australia.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Osborne LR, Mervis CB. Rearrangements of the Williams-Beuren syndrome locus: molecular basis and implications for speech and language development. Expert Rev Mol Med. 2007;9(15):1–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Antonell A, Del Campo M, Magano LF, Kaufmann L, de la Iglesia JM, Gallastegui F, Flores R, Schweigmann U, Fauth C, Kotzot D, et al. Partial 7q11.23 deletions further implicate GTF2I and GTF2IRD1 as the main genes responsible for the Williams-Beuren syndrome neurocognitive profile. J Med Genet. 2010;47(5):312–20.View ArticlePubMedGoogle Scholar
- Enkhmandakh B, Makeyev AV, Erdenechimeg L, Ruddle FH, Chimge NO, Tussie-Luna MI, et al. Essential functions of the Williams-Beuren syndrome-associated TFII-I genes in embryonic development. Proc Natl Acad Sci U S A. 2009;106(1):181–6.Google Scholar
- Lucena J, Pezzi S, Aso E, Valero MC, Carreiro C, Dubus P, Sampaio A, Segura M, Barthelemy I, Zindel MY, et al. Essential role of the N-terminal region of TFII-I in viability and behavior. BMC Med Genet. 2010;11:61.View ArticlePubMedPubMed CentralGoogle Scholar
- Young EJ, Lipina T, Tam E, Mandel A, Clapcote SJ, Bechard AR, Chambers J, Mount HT, Fletcher PJ, Roder JC, et al. Reduced fear and aggression and altered serotonin metabolism in Gtf2ird1-targeted mice. Genes Brain Behav. 2008;7(2):224–34.View ArticlePubMedGoogle Scholar
- Howard ML, Palmer SJ, Taylor KM, Arthurson GJ, Spitzer MW, Du X, et al. Mutation of Gtf2ird1 from the Williams-Beuren syndrome critical region results in facial dysplasia, motor dysfunction, and altered vocalisations. Neurobiol Dis. 2012;45(3):913–22.Google Scholar
- Schneider T, Skitt Z, Liu Y, Deacon RM, Flint J, Karmiloff-Smith A, Rawlins JN, Tassabehji M. Anxious, hypoactive phenotype combined with motor deficits in Gtf2ird1 null mouse model relevant to Williams syndrome. Behav Brain Res. 2012;233(2):458–73.View ArticlePubMedGoogle Scholar
- Tassabehji M, Hammond P, Karmiloff-Smith A, Thompson P, Thorgeirsson SS, Durkin ME, Popescu NC, Hutton T, Metcalfe K, Rucka A, et al. GTF2IRD1 in craniofacial development of humans and mice. Science. 2005;310(5751):1184–7.View ArticlePubMedGoogle Scholar
- Palmer SJ, Tay ES, Santucci N, Cuc Bach TT, Hook J, Lemckert FA, Jamieson RV, Gunnning PW, Hardeman EC. Expression of Gtf2ird1, the Williams syndrome-associated gene, during mouse development. Gene Expr Patterns. 2007;7(4):396–404.View ArticlePubMedGoogle Scholar
- Canales CP, Wong AC, Gunning PW, Housley GD, Hardeman EC, Palmer SJ. The role of GTF2IRD1 in the auditory pathology of Williams-Beuren Syndrome. Eur J Hum Genet. 2015;23(6):774–80.View ArticlePubMedGoogle Scholar
- Polly P, Haddadi LM, Issa LL, Subramaniam N, Palmer SJ, Tay ES, Hardeman EC. hMusTRD1alpha1 represses MEF2 activation of the troponin I slow enhancer. J Biol Chem. 2003;278(38):36603–10.View ArticlePubMedGoogle Scholar
- Vullhorst D, Buonanno A. Multiple GTF2I-like repeats of general transcription factor 3 exhibit DNA binding properties. Evidence for a common origin as a sequence-specific DNA interaction module. J Biol Chem. 2005;280(36):31722–31.View ArticlePubMedGoogle Scholar
- Issa LL, Palmer SJ, Guven KL, Santucci N, Hodgson VR, Popovic K, Joya JE, Hardeman EC. MusTRD can regulate postnatal fiber-specific expression. Dev Biol. 2006;293(1):104–15.View ArticlePubMedGoogle Scholar
- Palmer SJ, Santucci N, Widagdo J, Bontempo SJ, Taylor KM, Tay ES, Hook J, Lemckert F, Gunning PW, Hardeman EC. Negative autoregulation of GTF2IRD1 in Williams-Beuren syndrome via a novel DNA binding mechanism. J Biol Chem. 2010;285(7):4715–24.View ArticlePubMedGoogle Scholar
- O'Leary J, Osborne LR. Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice. PLoS One. 2011;6(8):e23868.View ArticlePubMedPubMed CentralGoogle Scholar
- Chimge NO, Mungunsukh O, Ruddle F, Bayarsaihan D. Expression profiling of BEN regulated genes in mouse embryonic fibroblasts. J Exp Zool B Mol Dev Evol. 2007;308(3):209–24.View ArticlePubMedGoogle Scholar
- Chimge NO, Makeyev AV, Ruddle FH, Bayarsaihan D. Identification of the TFII-I family target genes in the vertebrate genome. Proc Natl Acad Sci U S A. 2008;105(26):9006–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40.View ArticlePubMedGoogle Scholar
- Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11(10):R106.View ArticlePubMedPubMed CentralGoogle Scholar
- Peukert D, Weber S, Lumsden A, Scholpp S. Lhx2 and Lhx9 determine neuronal differentiation and compartition in the caudal forebrain by regulating Wnt signaling. PLoS Biol. 2011;9(12):e1001218.View ArticlePubMedPubMed CentralGoogle Scholar
- Rhee H, Polak L, Fuchs E. Lhx2 maintains stem cell character in hair follicles. Science. 2006;312(5782):1946–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Beer HD, Bittner M, Niklaus G, Munding C, Max N, Goppelt A, Werner S. The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene. 2005;24(34):5269–77.View ArticlePubMedGoogle Scholar
- Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. Am J Biochem. 2010;147(1):35–51.View ArticleGoogle Scholar
- Croce JC, McClay DR. Evolution of the Wnt pathways. Methods Mol Biol. 2008;469:3–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Doi T, Puri P, Bannigan J, Thompson J. Disruption of noncanonical Wnt/CA2+ pathway in the cadmium-induced omphalocele in the chick model. J Pediatr Surg. 2010;45(8):1645–9.View ArticlePubMedGoogle Scholar
- Ouji Y, Yoshikawa M, Shiroi A, Ishizaka S. Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells. Biochem Biophys Res Commun. 2006;342(4):1063–9.View ArticlePubMedGoogle Scholar
- Carmona-Mora P, Widagdo J, Tomasetig F, Canales CP, Cha Y, Lee W, Alshawaf A, Dottori M, Whan RM, Hardeman EC, et al. The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation. Hum Genet. 2015;134(10):1099–115.View ArticlePubMedGoogle Scholar
- Peterson LF, Boyapati A, Ranganathan V, Iwama A, Tenen DG, Tsai S, Tsai S, Zhang DE. The hematopoietic transcription factor AML1 (RUNX1) is negatively regulated by the cell cycle protein cyclin D3. Mol Cell Biol. 2005;25(23):10205–19.Google Scholar
- Zong H, Chi Y, Wang Y, Yang Y, Zhang L, Chen H, Jiang J, Li Z, Hong Y, Wang H, et al. Cyclin D3/CDK11p58 complex is involved in the repression of androgen receptor. Mol Cell Biol. 2007;27(20):7125–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Sarruf DA, Iankova I, Abella A, Assou S, Miard S, Fajas L. Cyclin D3 promotes adipogenesis through activation of peroxisome proliferator-activated receptor gamma. Mol Cell Biol. 2005;25(22):9985–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Despouy G, Bastie JN, Deshaies S, Balitrand N, Mazharian A, Rochette-Egly C, Chomienne C, Delva L. Cyclin D3 is a cofactor of retinoic acid receptors, modulating their activity in the presence of cellular retinoic acid-binding protein II. J Biol Chem. 2003;278(8):6355–62.View ArticlePubMedGoogle Scholar
- Jian Y, Yan J, Wang H, Chen C, Sun M, Jiang J, Lu J, Yang Y, Gu J. Cyclin D3 interacts with vitamin D receptor and regulates its transcription activity. Biochem Biophys Res Commun. 2005;335(3):739–48.View ArticlePubMedGoogle Scholar
- Weiss A, Attisano L. The TGFbeta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol. 2013;2(1):47–63.View ArticlePubMedGoogle Scholar
- Ichimura T, Watanabe S, Sakamoto Y, Aoto T, Fujita N, Nakao M. Transcriptional repression and heterochromatin formation by MBD1 and MCAF/AM family proteins. J Biol Chem. 2005;280(14):13928–35.View ArticlePubMedGoogle Scholar
- Sasai N, Saitoh N, Saitoh H, Nakao M. The transcriptional cofactor MCAF1/ATF7IP is involved in histone gene expression and cellular senescence. PLoS One. 2013;8(7), e68478.View ArticlePubMedPubMed CentralGoogle Scholar
- Piao L, Nakagawa H, Ueda K, Chung S, Kashiwaya K, Eguchi H, Ohigashi H, Ishikawa O, Daigo Y, Matsuda K, et al. C12orf48, termed PARP-1 binding protein, enhances poly(ADP-ribose) polymerase-1 (PARP-1) activity and protects pancreatic cancer cells from DNA damage. Genes Chromosomes Cancer. 2011;50(1):13–24.View ArticlePubMedGoogle Scholar
- Alshareeda AT, Negm OH, Green AR, Nolan CC, Tighe P, Albarakati N, et al. KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer. Br J Cancer. 2015;112(12):1929–37.Google Scholar
- Follit JA, San Agustin JT, Xu F, Jonassen JA, Samtani R, Lo CW, Pazour GJ. The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex. PLoS Genet. 2008;4(12), e1000315.Google Scholar
- Infante C, Ramos-Morales F, Fedriani C, Bornens M, Rios RM. GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein. J Cell Biol. 1999;145(1):83–98.View ArticlePubMedPubMed CentralGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.View ArticlePubMedGoogle Scholar
- Henrichsen CN, Csardi G, Zabot MT, Fusco C, Bergmann S, Merla G, Reymond A. Using transcription modules to identify expression clusters perturbed in Williams-Beuren syndrome. PLoS Comput Biol. 2011;7(1), e1001054.View ArticlePubMedPubMed CentralGoogle Scholar
- O'Mahoney JV, Guven KL, Lin J, Joya JE, Robinson CS, Wade RP, Hardeman EC. Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1. Mol Cell Biol. 1998;18(11):6641–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Tantin D, Tussie-Luna MI, Roy AL, Sharp PA. Regulation of immunoglobulin promoter activity by TFII-I class transcription factors. J Biol Chem. 2004;279(7):5460–9.View ArticlePubMedGoogle Scholar
- Yan X, Zhao X, Qian M, Guo N, Gong X, Zhu X. Characterization and gene structure of a novel retinoblastoma-protein-associated protein similar to the transcription regulator TFII-I. Biochem J. 2000;345(Pt 3):749–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Tussie-Luna MI, Bayarsaihan D, Seto E, Ruddle FH, Roy AL. Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxbeta. Proc Natl Acad Sci U S A. 2002;99(20):12807–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Folgueras AR, Guo X, Pasolli HA, Stokes N, Polak L, Zheng D, Fuchs E. Architectural niche organization by LHX2 is linked to hair follicle stem cell function. Cell Stem Cell. 2013;13(3):314–27.View ArticlePubMedGoogle Scholar
- Mardaryev AN, Meier N, Poterlowicz K, Sharov AA, Sharova TY, Ahmed MI, Rapisarda V, Lewis C, Fessing MY, Ruenger TM, et al. Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury. Development. 2011;138(22):4843–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Birk E, Har-Zahav A, Manzini CM, Pasmanik-Chor M, Kornreich L, Walsh CA, Noben-Trauth K, Albin A, Simon AJ, Colleaux L, et al. SOBP is mutated in syndromic and nonsyndromic intellectual disability and is highly expressed in the brain limbic system. Am J Hum Genet. 2010;87(5):694–700.View ArticlePubMedPubMed CentralGoogle Scholar
- Godoy JA, Rios JA, Zolezzi JM, Braidy N, Inestrosa NC. Signaling pathway cross talk in Alzheimer's disease. Cell Commun Signal. 2014;12:23.View ArticlePubMedPubMed CentralGoogle Scholar
- Collu GM, Hidalgo-Sastre A, Brennan K. Wnt-Notch signalling crosstalk in development and disease. Cell Mol Life Sci. 2014;71(18):3553–67.View ArticlePubMedGoogle Scholar
- Katoh M, Katoh M. Cross-talk of WNT and FGF signaling pathways at GSK3beta to regulate beta-catenin and SNAIL signaling cascades. Cancer Biol Ther. 2006;5(9):1059–64.View ArticlePubMedGoogle Scholar
- Itoh N, Ornitz DM. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease. J Biochem. 2011;149(2):121–30.View ArticlePubMedGoogle Scholar
- Turner N, Grose R. Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer. 2010;10(2):116–29.View ArticlePubMedGoogle Scholar
- Yun YR, Won JE, Jeon E, Lee S, Kang W, Jo H, Jang JH, Shin US, Kim HW. Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng. 2010;2010:218142.View ArticlePubMedPubMed CentralGoogle Scholar
- van Scheltinga AF T, Bakker SC, Kahn RS, Kas MJ. Fibroblast growth factors in neurodevelopment and psychopathology. Neuroscientist. 2013;19(5):479–94.View ArticleGoogle Scholar
- Stevens HE, Smith KM, Maragnoli ME, Fagel D, Borok E, Shanabrough M, Horvath TL, Vaccarino FM. Fgfr2 is required for the development of the medial prefrontal cortex and its connections with limbic circuits. J Neurosci. 2010;30(16):5590–602.View ArticlePubMedPubMed CentralGoogle Scholar
- Yamanaka Y, Kitano A, Takao K, Prasansuklab A, Mushiroda T, Yamazaki K, Kumada T, Shibata M, Takaoka Y, Awaya T, et al. Inactivation of fibroblast growth factor binding protein 3 causes anxiety-related behaviors. Mol Cell Neurosci. 2011;46(1):200–12.View ArticlePubMedGoogle Scholar
- Szebenyi G, Fallon JF. Fibroblast growth factors as multifunctional signaling factors. Int Rev Cytol. 1999;185:45–106.View ArticlePubMedGoogle Scholar
- Bikle DD, Xie Z, Tu CL. Calcium regulation of keratinocyte differentiation. Expert Rev Endocrinol Metab. 2012;7(4):461–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim J, Kim J, Kim DW, Ha Y, Ihm MH, Kim H, Song K, Lee I. Wnt5a induces endothelial inflammation via beta-catenin-independent signaling. J Immunol. 2010;185(2):1274–82.View ArticlePubMedGoogle Scholar
- Flavell SW, Greenberg ME. Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu Rev Neurosci. 2008;31:563–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Rosso SB, Inestrosa NC. WNT signaling in neuronal maturation and synaptogenesis. Front Cell Neurosci. 2013;7:103.View ArticlePubMedPubMed CentralGoogle Scholar
- Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E. Oxytocin increases trust in humans. Nature. 2005;435(7042):673–6.View ArticlePubMedGoogle Scholar
- Dai L, Carter CS, Ying J, Bellugi U, Pournajafi-Nazarloo H, Korenberg JR. Oxytocin and vasopressin are dysregulated in Williams Syndrome, a genetic disorder affecting social behavior. PLoS One. 2012;7(6), e38513.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36.View ArticlePubMedPubMed CentralGoogle Scholar
- Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11(3):R25.View ArticlePubMedPubMed CentralGoogle Scholar
- Saito R, Smoot ME, Ono K, Ruscheinski J, Wang PL, Lotia S, Pico AR, Bader GD, Ideker T. A travel guide to Cytoscape plugins. Nat Methods. 2012;9(11):1069–76.View ArticlePubMedPubMed CentralGoogle Scholar
- Supek F, Bosnjak M, Skunca N, Smuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One. 2011;6(7), e21800.View ArticlePubMedPubMed CentralGoogle Scholar
- Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50.View ArticlePubMedPubMed CentralGoogle Scholar