The zebrafish skull transitions from primarily cartilage (blue) in larvae to primarily bone (magenta) in adults.
Preventing Precocious Differentiation During Craniofacial Development
Mouse (left) and zebrafish (right) embryos differ tremendously in size but use the same set of genes to build their skull.
Both organ development and homeostasis require that the differentiation of progenitors into mature cell types be balanced with some level of progenitor maintenance or self-renewal. This ensures that sufficient uncommitted precursors will be available to generate later-differentiating cell types as well as long-term adult stem cells.
How, then, do certain progenitor cells elude early differentiation in developing tissues? Some cells may simply be too distant from a source of pro-maturation signals to initiate a differentiation program. However, the complexity of differentiation patterns implies that additional active mechanisms may also be at play.
The skull is a prime example of a complex organ composed of multiple cell types that all derive from cranial neural crest cells but differentiate at different times during development. Cartilage is the first mature cell type to appear, with intramembranous bones and connective tissues developing later.
Are some facial progenitor cells programmed to resist early cartilage-inducing cues to ensure the later availability of bone and connective tissue precursors? In support of this idea, we have identified a series of zebrafish mutants that show excess cartilage formation and depletion of bone where the mutated genes are normally expressed in progenitors. Intriguingly, overexpression of the same genes prevents progenitors from becoming cartilage but does not lead to excess bone, implying that they are not merely “pro-bone” factors. Instead, these genes appear to act intrinsically within progenitors to block their early differentiation into cartilage, overriding any pro-differentiation signals they may encounter.
To further investigate these questions, we are using both zebrafish and mouse models to perform lineage tracing, transplantation, transcriptional profiling, and chromatin-binding assays, as well as applying CRISPR/Cas9 technology to generate new reporter and mutant alleles for analysis. Genes of special interest include the Nr2f nuclear receptors, components of the Notch pathway, and the Prrx1 transcription factors.
We have additional evidence that some of these same genes are also involved in the initial specification of facial skeletal progenitors within the cranial neural crest and/or in the maintenance of bone stem cells in adults. Additional experiments will determine the onset and duration of the requirement for these genes in facial progenitors and more generally in neural crest cells. By studying this group of genes in parallel, we hope to unravel the genetic and molecular mechanisms that confer, protect, and maintain stemness throughout the lifetime of a skeletal progenitor cell.
Complex patterns of gene expression in the pharyngeal arches give rise to complex skeletal shapes in the skull.
Screening New Candidate Craniofacial Genes
Dozens of studies have demonstrated that the genes and cellular processes that build the skull are highly conserved across vertebrate species, meaning that we can learn a great deal about the genetics of mammalian skull development using the more tractable zebrafish model. We have on hand a set of ~40 zebrafish mutant lines for genes classified as confirmed or candidate regulators of craniofacial development. New lines for candidate genes identified through exome or genome sequencing of patients at Cincinnati Children’s are also now being generated using CRISPR / Cas9 technology. In addition, we are always on the lookout for genes expressed in restricted patterns in the developing face, which can indicate a specific role for that gene in the formation of a particular skeletal element.
In addition to ascertaining the phenotype of each new line, we investigate the consequences of different combinations of mutations to determine whether the genes function additively, synergistically, or antagonistically. Zebrafish provide a powerful model for these kinds of complex genetic studies, as one pair of fish can generate approximately a hundred offspring per week for months on end, allowing analysis of not only double and triple mutant combinations but also quadruples and quintuples. By determining whether and how our candidate genes contribute to skull formation in fish, we hope to provide improved genetic diagnoses for patients affected by disorders of craniofacial development.