Student Theses and Dissertations
Investigation into the Neurogenetics of Vocal Learning in Tursiops Truncatus, the Bottlenose Dolphin
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Erich D. Jarvis
Additional Thesis Advisor
Marcelo O. Magnasco
Keywords
vocal learning, cetacean neurogenetics, bottlenose dolphin, motor cortex, genome assembly, single-nuclei RNA sequencing (snRNA-seq)
Abstract
Vocal learning, a necessary substrate for speech, is a rare trait found in only five mammalian and three avian lineages. Each of these vocal learning groups is distantly related to one another, and each has a close non-learning relative, indicating that the trait evolved independently in each lineage rather than being present in a common ancestor. Studies in songbirds and humans have demonstrated that functionally analogous forebrain regions within this circuit exhibit specialized gene expression profiles, distinguishing them from the surrounding circuits. Although cetaceans are among the most advanced mammalian vocal learners, the underlying brain circuitry of this behavior remains unknown. By studying the neurogenetics of vocal learning in intelligent mammals, we can gain a deeper understanding of the evolution of spoken language. As a first step in investigating the neurogenetic underpinnings of cetacean vocal learning, we generated a haplotype-phased chromosome-scale reference genome assembly for the bottlenose dolphin (Tursiops truncatus). Bottlenose dolphins are well-documented learners that share significant phenotypic overlap with human language development. Using a trio binning approach developed by the Vertebrate Genome Project (VGP), in which samples from an individual and both parents are sequenced, we generated a fully phased diploid assembly in which both haplotypes were assembled in their entirety. The use of multiple long-read technologies allowed for the generation of a highly intact and accurate genome, which significantly surpassed the quality of previously published assemblies for this species. This genome is now the National Center for Biotechnology Information (NCBI) reference assembly. Building on the success of this work, we co-founded the Cetacean Genome Project (CGP) with the National Oceanic and Atmospheric Administration (NOAA), an initiative aimed at generating comparably high-quality reference genomes for all cetaceans. To make gene expression characterization experiments feasible in dolphins, we first needed to overcome the challenge of collecting and preserving high-quality brain samples from necropsies of stranded dolphins. To this end, we developed a novel brain extraction protocol in which the brain was removed fully intact and preserved in the field, with the left hemisphere placed in chilled formalin and the right hemisphere flash-frozen on dry ice. We utilized this protocol in collaboration with the NOAA stranding response network to opportunistically collect specimens from necropsy procedures. In this study, we demonstrated the feasibility of this methodology for collecting high-quality samples. We expanded this methodology to collect samples from other cetacean species, establishing the North American Cetacean Brain Bank. With this collection of high-quality frozen samples, we investigated whether the dolphin motor cortex exhibits a region with specialized gene expression, similar to that shared between the human laryngeal motor cortex (LMC) and functionally analogous vocal learning nuclei in songbirds (HVC and RA). To screen for the putative “vocal learning” specialized subregion of the identified dolphin motor cortex, we conducted double-label Fluorescent in Situ Hybridization (FISH) experiments for SLIT1, a downregulated marker for these regions, and parvalbumin (PVALB), an upregulated marker. Standard FISH protocols are typically designed for small laboratory specimens. We have developed a large tissue protocol that enables the imaging of whole hemispheres of human and dolphin brains. The brains were sectioned, tape-transferred, and embedded in nitrocellulose. Utilizing this technique, we identified a region of the bottlenose dolphin motor cortex that exhibits the PVALB upregulation and SLIT1 downregulation signatures observed in the LMC and HVC/RA of humans and songbirds. Using the FISH images as a reference point, we dissected samples from the specialized region identified in the dolphin motor cortex and performed single-nuclei RNA sequencing (snRNA-sequencing) of these regions. Initial cell typing revealed that an overwhelming proportion of these cells were glial cells. Although further work is required to fully characterize the transcriptomic identity of this cetacean brain, this body of work provides initial support for the existence of an LMC-analogous region in bottlenose dolphins.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Maloney, Brigid, "Investigation into the Neurogenetics of Vocal Learning in Tursiops Truncatus, the Bottlenose Dolphin" (2025). Student Theses and Dissertations. 815.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/815
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy