Transcriptomic analysis reveals the role of SIX1 in mouse cranial neural crest patterning and bone development

Abstract

Background: Genetic variants of the transcription factor SIX1 and its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is characterized by craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. In this work, we expand our knowledge of the Six1 gene regulatory network by using a Six1-null mouse line to assess gene expression profiles of E10.5 mandibular arches, which give rise to the neural crest (NC)-derived middle ear ossicles and lower jaw, via bulk RNA sequencing.

Results: Our transcriptomic analysis led to the identification of 808 differentially expressed genes that are related to translation, NC cell differentiation, osteogenesis, and chondrogenesis including components of the WNT signaling pathway. As WNT signaling is a known contributor to bone development, we demonstrated that SIX1 is required for expression of the WNT antagonist Frzb in the mandibular arch, and determined that SIX1 expression results in repression of WNT signaling.

Conclusion: Our results clarify the mechanisms by which SIX1 regulates the development of NC-derived craniofacial elements that are altered in SIX1-associated disorders. In addition, this work identifies novel genes that could be causative to this birth defect and establishes a link between SIX1 and WNT signaling during patterning of NC cells.

Keywords: Branchio-oto-renal syndrome; SIX1; craniofacial defects; hearing loss; transcriptional regulation.

Figure 1:. Workflow for comparative bulk RNA-seq analysis of wild type, Six1-het and Six1-null mandibular arches.

(A–C) OPT images of wild type (WT), Six1-het (HET), and Six1-null (NULL) mouse heads reveal that, in addition to deformed skull and jaws, Six1-null mice present with BOR-like features such as ear tags (arrow in C). (D) Expression pattern for Six1 as seen with whole-mount ISH in WT E10.5 mouse embryo. Mandibular arches (dashed yellow box) were manually dissected and processed for RNA-seq. Ventral view. (E) Venn diagram showing the number of genes detected in each genotype. (F) Quantitative expression of Six1 and reference gene Gapdh in mandibular arches from each genotype. n=3 per genotype. Data was normalized with expression values for Actb. **P<0.01, ****P<0.0001, ns: not significant.

Figure 2:. Differential expression analysis of genes in WT, Six1-het and Six1-null mandibular arches.

(A) Heatmap with K-means cluster analysis of averaged, z-score scaled, FPKM values for gene expression in each genotype. Yellow bars mark cluster number. Grey boxes list gene ontology terms represented in each cluster. (B) Quantitative-PCR (qPCR) validation of expression pattern of select genes from cluster 1 (Bgn), 5 (Frzb), 6 (Lum, Tubb3), 7 (Alx1, Fgf9) in WT, Six1-het (HET) and Six1-null (NULL) arches. *P<0.05, ** P< 0.01, *** P<0.001, **** P<0.0001, ns: not significant. n=3. Actb was used as housekeeping gene.

Figure 3:. Comparison of differentially expressed genes with BOR associated phenotypes as described in OMIM and MGI databases.

(A) Venn diagram showing differentially expressed genes that have been associated with ear/hearing and craniofacial anomalies. (B–C) Volcano plots marking differentially expressed genes between WT and Six1-null (NULL) arches in blue (FDR ≤ 0.25, i.e. −0.6 on a log₁₀ scale). Data points marked in red represent genes that have been associated with ear/hearing anomalies and craniofacial anomalies.

Figure 4:. Regulation of WNT signaling by SIX1.

(A-C) Whole-mount ISH for Frzb in E10.5 WT (A), Six1-het (HET) (B), and Six1-null (NULL) (C) mouse embryos. Ventral view. The heart was removed to aid visualization. 1. Mandibular arch. Loss of Six1 disrupts expression of Frzb in the mandibular arch (arrow). (D-E) Luciferase activity of the TOP-Flash reporter vector is significantly decreased in MC3T3-E1 (D) and O9-1 (E) cells after Six1 overexpression indicating that SIX1 represses canonical WNT-specific transcriptional activity. ***P<0.001, ****P<0.0001. n=3.