Using Zebrafish as an in vivo Model for Frontonasal Development and Disease - Project Summary/Abstract
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.
