We study craniofacial development and the genetic causes of craniofacial birth defects.

Our goal is to determine the role of the transcription factor SIX1 and SIX1-associated genes in development of the cranial bones, jaws and ear using transgenic mice and Xenopus (frog).

Our overarching goal is to help patients and their families understand the genetic causes of SIX1-related birth defects (e.g., branchio-oto-renal syndrome, craniosynostosis), and to develop preventive and corrective strategies.

We are now looking for students, staff scientist (limited term researcher) and a postdoc! (look here)

Current Projects

1. Analyzing the function of the Six1 transcriptional complex (Six1+co-factors) during development of the middle ear and lower jaw cartilage and bone (Xenopus and mouse)

EDNRA signaling is required for patterning of neural crest cells that differentiate into cartilage and bone of the lower jaw and middle ear in the mouse first pharyngeal arch. Six1-null embryos present hypoplastic lower jaw and middle ear ossicles and deformed upper jaw. Because Six1 transcriptional function relies on interactions with co-factors, we are characterizing novel Six1 binding partners previously identified in yeast two-hybrid screens (fly and frog). To characterize function in vivo, we are employing the 2 models with which I have expertise: mouse and Xenopus. Xenopus is being used to study the function of novel binding partners (potential co-factors) during inner ear and craniofacial cartilage development. Translation-blocking morpholinos and CRISPR/Cas technology are being used to disrupt expression of these potential co-factors. Gene expression changes are being detected by ISH and qPCR, and the craniofacial phenotypes by cartilage staining and by using transgenic reporter lines. Xenopus can give rapid answers about the role of these factors in craniofacial development. To characterize the role of these novel co-factors during middle ear and jawbone development in mouse, we are utilizing available knockout mouse lines for these co-factors (KOMP, Jackson labs). We are analyzing these lines by verifying cartilage & bone structure, and expression pattern in the E10.5 first pharyngeal arch for genes regulated by EDNRA signaling and novel genes identified in my previous R03 grant supported RNA-seq study. Genetic interaction with Six1 will be verified by crossing these lines with the Six1-null line. Experiments will demonstrate if the identified binding partners have a role in inner ear, middle ear and jaw formation, and whether, as Six1 does, they regulate the expression of genes that are part of the EDNRA signaling which would indicate a potential link to craniofacial birth defects.

2. Determining the role of Six1 and its co-factors during calvarial bone and suture development (mouse)

Variants in the transcription factor SIX1 or its co-factor EYA1 are known underlying genetic causes of Branchio-oto-renal syndrome (BOR), an autosomal dominant disease that results in hearing loss and kidney defects. Recently, a clinical study reported premature cranial suture closure (craniosynostosis, CS) in individuals carrying SIX1 BOR variants, including 5’ variants (p.Q11X and p.Q22X) that are predicted to lead to haploinsufficiency, and variants that disrupt protein function (p.R110W); these data suggest that CS may be an undiagnosed defect in BOR. If left untreated, CS can be associated with distortion of skull shape, increased intracranial pressure, and/or brain damage. As defects in the calvarial bone osteoprogenitor cells (OPC) before and/or after birth may lead to CS via increased bone deposition in the cranial sutures, in this project, we plan to address major knowledge gaps regarding Six1 function: What is its role in the development of the calvarial bones?; What is its role in the pathophysiology of CS? To address these questions, we are using transgenic mouse lines (Six1 knockout line, a line carrying the pR110W mutation (Six1pR110W), a conditional Six1 knockout line (Six1fl/fl). To detect changes in bone development and morphology, we are quantitatively analyzing head morphology using µCT images and tissue formation using histological analyses. To verify if Six1 and its co-factors have a role the specification and differentiation of OPCs, we are assessing gene expression in vivo using RNAscope/HCR and qPCR and in vitro using neural crest-derived mesenchymal precursors and OPCs. Lastly, we will perform single cell RNA-seq and RNAscope to identify cell populations in the supraorbital arch mesenchyme (that gives rise to the rudiments for parietal and frontal bones) and in the cranial sutures that are affected by altering Six1 dosage and function. Results from this application will shift the paradigm of Six1 function as a cranial placode, neural and muscle transcriptional factor by providing the first direct evidence linking it to normal calvarial development and the pathogenesis of CS.

3. Dissecting the function of SIX1 and SIX1 co-factors during incisor development (mouse)

Odontogenesis involves a series of inductive interactions between the neural crest-derived odontogenic mesenchyme and the overlying ectoderm (dental lamina) of the first pharyngeal arch. Six1, Eya1, Sobp, Mcrs1 and Pa2g4 are expressed in the mouse odontogenic mesenchyme at E10.5. Preliminary data show that loss of Six1 disrupts expression of Shh in the lower incisor dental lamina at E12.5, and by E18.5 Six1-null embryos present no lower incisors. These data indicate that Six1 has a novel function in odontogenesis.

In this project, we will first determe at which stage incisor development is arrested using histological and molecular marker analyses of E10.5 to E16.5 Six1-/- mouse embryos. We will assess by ISH and qPCR the expression of odontogenic genes selected based on the literature and on the data obtained from my R03 grant supported RNA-Seq study. Because Six1+/- embryos still develop lower incisors, we will cross the Six1-null line with co-factor lines and the Eya1-null line and verify if loss of Six1 co-factor alleles in a Six1+/- background completely disrupts incisor development. Six1 conditional knockout will use to specifically address which Six1+ cell types contribute to the incisor phenotype. This work will establish Six1 as a novel regulator of lower incisor development and determine which co-factors are required for this function.