In this project, I studied the development of pharynx and head using various vertebrate models, mainly the fish species representing early divergent lineages bichir, sturgeon, and gar and transferred my knowledge and experience obtained from my work on jawless fish lamprey and other vertebrates to the host laboratory. We built an important genomic resource from the transcriptomes of several developmental stages of all three abovementioned fish species that has been subsequently used in designing tools for descriptive and functional genetic analyses. I introduced and troubleshot the CRISPR/Cas9 method of targeted mutagenesis in sturgeon, which allowed us to expand and detail our functional analyses using mainly pharmacological perturbances. Results of my work were used in the grant proposal of my host PI that has been awarded funding in 2018 and will serve as a basis for my own grant proposal that is planned to be submitted in early 2020.
The descriptive and functional analyses performed during the research showed that pharyngeal development of early divergent fishes is generally largely conserved among them as well as with their vertebrate relatives. However, clear and very exciting differences can be observed in timing of formation of some elements, such as bichir mandibulo-hyoid pharyngeal pouch and hyoid arch, which disrupts the general dogma of strictly antero-posterior pouch formation progression and might have played critical role in the evolution of the lineage specific novelties, such as bichir external gills, sturgeon rostrum, and gar and other vertebrate opercula. Furthermore, we found that the enigmatic transient pharyngeal structure, the pre-oral gut, of basal fishes clearly shares traits with pharyngeal pouch formation in its development as is evidenced by both shared genes expressed during their development and functional involvement of some genetic pathways such as FGFs and Retinoic acid. However, details in the expression pattern and targeted mutagenesis using CRISPR/Cas9 system also indicate fundamental differences between these structures since mutation of several key players of pharyngeal pouch formation (Eya1, Foxq1, Pax9, Ripply3) resulted in virtually no effect on the preoral gut development. To decipher the key players in preoral gut vs. pharyngeal pouch development, further study involving precise transcriptomic profiling of the specific cells and tissues will be necessary before the roles of identified genes can be tested functionally. The importance of this next step is highlighted by the fact that developmental pathways identified to underlie the preoral gut formation so far, such as FGFs, Shh, and Retinoic acid, have strong pleiotropic effects on neighboring tissues. Their inhibition, whether pharmacological or CRISPR/Cas9 mediated, prevents thus identification of the precise role preoral gut plays in the head skeleton patterning and development.
