Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - Pharynx2016 (Evolution and development of pharyngeal pouches and pre-oral gut in basal fishes)
Teaser
The origin of the phenomenal diversity of vertebrates lies in the transition of their life style from small filter feeding to large actively foraging creatures. This evolutionary event was enabled by modifications to the pharynx of our chordate ancestors leading to the...
Summary
The origin of the phenomenal diversity of vertebrates lies in the transition of their life style from small filter feeding to large actively foraging creatures. This evolutionary event was enabled by modifications to the pharynx of our chordate ancestors leading to the evolution of sophisticated head skeleton.
In this project I used developmental-genetic approach in phylogenetic comparative framework (evo-devo) to study the structural and developmental changes in pharyngeal evolution in early vertebrate divergences, namely around the split of jawed and jawless fishes and in the diversification of early bony fishes. Specifically, I focused on understanding the similarities and differences in the formation of various parts of pharynx and related tissues, such as pharyngeal pouches, preoral gut, and neural crest, and their importance in the evolution of vertebrate head skeleton.
The integral part of the project was two-way knowledge transfer between me and the host laboratory in an attempt to synergize our experience in embryology and functional genetics and generate resources for future evolutionary developmental research.
I believe the project has achieved its main objectives, significantly improved my experience and competence, and helped me to prepare for the role of an independent scientist and team leader.
Work performed
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.
Final results
The project generated useful genomic resources of the phylogenetically important vertebrate species that can be used in further studies as well as by other researchers in genomic, developmental, and evolutionary research. I also introduced the method of targeted mutagenesis to a commercially important fish sturgeon and its application may have important socioeconomic consequences in future. The main scientific results are in the realm of basic evolutionary and developmental research and contributed important information to our understanding of the pharyngeal and head skeleton evolution of vertebrates.