Barske Lab

Projects

Making Skeletal Progenitors for the Skull

Craniofacial defects of the most severe and lethal type are caused by aberrations at early stages of facial development. One early event that can go awry is the establishment of the skeletal progenitor pool that will build the face. These cells derive from a subpopulation of cranial neural crest cells (CNCCs) that transition from a migratory neural crest state to a skeletal progenitor state upon their arrival at the pharyngeal arches. All subsequent stages of craniofacial development depend on this transition occurring successfully.

A deficiency of skeletal progenitors can lead to no facial skeleton forming at all (in extreme cases) or to mildly dysmorphic features, depending on the extent of the loss. However, the mechanisms guiding this transition remain obscure.

We are using mouse and zebrafish models to test the hypothesis that the NR2F family of nuclear receptors work redundantly and cell-autonomously to activate the skeletal progenitor state in CNCCs. This would be not only a new function for these key developmental regulators, but also a new entry point into the longstanding puzzle of how facial progenitors acquire their skeletal potency.

Bone mineralization in adult vertebrates is controlled by a complex balance of endocrine hormones. However, which hormones and endocrine organs are critical for the initiation of mineralization in the embryo is a gap in knowledge.

We have identified a mutant zebrafish line with severely deficient mineralization of unknown etiology. The mutated gene is a human disease gene that is very important for neural crest development but has not previously been linked to skeletal development.

We are testing that the systemic mineralization deficit is due to a deficient neural crest function or contribution to a critical endocrine organ. Tracing the responsible organ will provide new insight into neural crest-dependent signals that trigger mineralization of the skeleton.

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.

Evolution of Skeletal Patterning across Vertebrates

We are also interested in how spatiotemporal shifts in gene expression during development underlie changes in the vertebrate body plan. In 2020, we reported Pou3f3 as an essential gene for fish gill cover development whose expression correlates with the diversity of pharyngeal appendages (or lack thereof) that form in lamprey, skates, elephant fish, and mammals.

Now, branching into the post-cranial skeleton, we are studying another zebrafish mutant with a remarkable midline fin phenotype that is providing fresh insight into how appendages are positioned along the body axis.