In humans, mutations in the aristaless-like homebox 3 (ALX3) transcription factor encoding gene are linked to the autosomal recessive disorder frontonasal dysplasia (FND). FND phenotypes include midline neurocranium defects like dysmorphic frontal, maxillary and ethmoid bones, along with soft tissue defects like wide and short nasal bridge, and bifid nasal tip. Yet, mouse Alx3 mutants in an otherwise wild-type background do not display FND phenotypes. Therefore, there is no animal model for ALX3 linked FND. However, genetic control of the zebrafish craniofacial skeleton is similar to mammals. Thus, I propose that the zebrafish model will fill the gap in our understanding of this genetic disease. Specifically, we generated an alx3 loss of function mutant allele with neurocranium phenotypes consistent with those seen in humans. My preliminary results show that the zebrafish system is suitable for this research. Transcriptomic and in situ gene expression analysis show that the alx3 gene is specifically expressed in frontonasal neural crest cells (fNCC), the progenitor cells of the frontonasal skeleton in the neurocranium. CRISPR/Cas9 mutagenesis demonstrates that alx3 function is required for proper development of the frontonasal skeleton elements like the ethmoid plate, and the parasphenoid bone. Like humans, zebrafish alx3 mutants develop midline skeletal defects. Specifically, homozygous alx3 mutants display (1) an excess of cartilage at the posterior ethmoid plate, (2) loss of cellular organization in the ethmoid plate, and (3) a blunted parasphenoid bone. In this proposal I will test the hypothesis that alx3 cell-autonomously functions in frontonasal neural crest cells to control cellular identity in the zebrafish neurocranium within a conserved genetic network. I will directly test this hypothesis by examining alx3 expression and function with cellular resolution expression studies and cellular transplantations (AIM1), by determining how alx3 controls cell fate with lineage tracing (AIM2), and by analyzing if alx3 functions in a genetic network conserved between fish and mammals during neurocranium development (AIM3). The results collected from this proposal will form the foundation for my long-term objective of understanding frontonasal development in general and the origin of pathogenesis of human FND linked to ALX3. This work will potentially inform and improve medical practices including genetic counseling, diagnosis and disease management. This proposal contains a training plan that will provide me with both scientific training and career development opportunities to support the intention of PA-19-188, to promote engagement of under-represented minorities as independent investigators in health-related research. This plan includes course work, seminars, attending local, regional and national meetings, sponsor and co- sponsor path correction meetings, a mentoring committee with internal and external members that will oversee my transition to independence, and access to multiple research and career development resources available only at the University of Colorado - Anschutz Medical campus and the Department of Craniofacial Biology.
This study of frontonasal development during normal embryonic development and in a disease model informs our understanding of the congenital craniofacial birth defect frontonasal dysplasia (FND). Over the last decade, there has been no progress in our knowledge of ALX3-related human FND. Developing a zebrafish model of this disease will allow progress toward treatment of this disease. This project establishes the foundation for a future research line where I will develop an independent research career addressing the genetic, cellular and developmental mechanism of FND. This plan is in line with NIH?s mission to seek fundamental knowledge that will help to reduce and prevent human disabilities.
