# Identification of SIX1-related genes as potential candidates for craniofacial birth defects

> **NIH NIH R03** · GEORGE WASHINGTON UNIVERSITY · 2020 · $159,500

## Abstract

ABSTRACT
Many congenital craniofacial disorders result from abnormal regulation of cranial neural crest cell (NCC)
formation, migration or patterning within the mandibular portion of pharyngeal arch 1 (mandibular arch). The
transcription factor SIX1 is critical for craniofacial development with mutations in SIX1 and its co-factor EYA1
identified as the underlying genetic causes of around 50% of Branchiootorenal spectrum disorder (BOS) cases.
BOS is an autosomal dominant disorder, in which affected individuals that have one normal allele present
variable degrees of craniofacial defects, hearing impairment, and renal abnormalities. Preliminary data from
mice demonstrate that decreased levels of Six1 in heterozygotes (Six1+/-) affect the expression of four genes
related to craniofacial development (Gbx2, Dlx3, Dlx2 and Hand2) more significantly than complete Six1 loss
(Six1-/-); and that known (Eya1, Eya2) and putative (Pa2g4, Mcrs1, Sobp) co-factors are expressed in distinct
regions of the mandibular arch with Six1. Thus, it is possible that normal dosage of Six1 regulates
morphogenesis of multiple NCC-derived structures by inducing or repressing downstream genes, and that
these transcriptional activities are the consequence of interactions with different levels of co-factors in distinct
domains of the mandibular arch. In this R03 application I aim to identify: 1) SIX1-regulated genes within two
specific mandibular arch domains; and 2) the functional interactions between SIX1 and putative co-factors
PA2G4, MCRS1 and SOBP. These goals will be addressed in two specific aims. In Aim 1, genes that require
proper Six1 dosage will be identified in the ventral plus intermediate domains of the mandibular arch. RNA-seq
of dissected domains of the arch from Six1 heterozygotes and Six1-nulls will provide a list of genes that require
normal levels of Six1. The top 10 most significantly affected genes from each genotype will be validated by
quantitative real-time PCR and whole-mount and sectional RNAscope In Situ Hybridization (ISH). In Aim 2, the
expression patterns of potential co-factors PA2G4, MCRS1 and SOBP will be characterized in relation to that
of SIX1 in mouse embryos by dual probe whole-mount ISH and Immunofluorescence microscopy. Putative co-
factors that overlap with SIX1 will be assayed in the NCC line O9-1 for whether they bind to SIX1, modulate
SIX1 transcriptional activity and regulate the expression of SIX1-regulated genes from Aim 1. Results will
provide a deeper understanding of SIX1 function and ultimately provide the key to understanding the
underlying genetics of the remaining BOS cases or other craniofacial disorders. Validated SIX1-regulated
genes and identified co-factors will be studied in future R01 applications in relation to BOS and other
craniofacial disorders.

## Key facts

- **NIH application ID:** 9960496
- **Project number:** 5R03DE028964-02
- **Recipient organization:** GEORGE WASHINGTON UNIVERSITY
- **Principal Investigator:** Andre L P Tavares
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $159,500
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9960496

## Citation

> US National Institutes of Health, RePORTER application 9960496, Identification of SIX1-related genes as potential candidates for craniofacial birth defects (5R03DE028964-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9960496. Licensed CC0.

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