Multi-omics Analysis Reveals the Development of Bat High-Frequency Hearing and the Evolution History of Laryngeal Echolocation

Abstract

Bats are unique among mammals, possessing remarkable adaptations including true self-powered flight, laryngeal echolocation (LE), exceptional longevity, unique immunity, and vocal learning. Among these, their unique form of LE allows them to navigate in total darkness using sound alone, marking a distinctive presence in the evolutionary history of life. However, the evolutionary history of LE in bats remains unresolved. Since bats with LE have the capability of high-frequency hearing, studying their auditory systems has become a crucial approach to unravel the evolutionary history of LE. However, so far, the understanding of the bat hearing system is far from sufficient, and more data is urgently needed to reveal a more comprehensive figure. In this thesis, I utilize advanced bioinformatics approach to analyze bat genomic and transcriptomic data, providing new insights into the evolutionary history of the bat echolocation system and shedding light on the molecular differences in hearing mechanisms among different species.

Reference genomes are crucial resources in evolutionary biology, and obtaining high-quality genomes is essential for understanding the molecular basis and evolution of LE. In this thesis, we initially concentrated on generating high quality reference-quality genomes for two Asian LE bat species, Vespertilio sinensis and Rhinolophus cornutus. To achieve this, we employed a combination of Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C scaffolding techniques. We obtained high quality chromosome-level genome, with 31 chromosomes clusters in Rhinolophus cornutus and 19 chromosomes clusters in Vespertilio sinensis. Genome synteny analysis reveal that bat exhibit high genomic synteny on X chromosome. Subsequently, we annotated or reannotated an additional 14 bat species and combined these with four outgroup mammals to create a comparative genome database. Phylogenetic analysis confirmed that the Pteropodidae family is the sister group to Rhinolophoidea, and Yangochiroptera constitutes a monophyletic group. The positive selection test highlighted the divergent molecular mechanisms associated with echolocation between Yangochiroptera and Rhinolophoidea, which involved different genes under positive selection related to echolocation. In contrast, Pteropodidae exhibited no genes under positive selection linked to vocal learning. Furthermore, 8 of the 13 positive selection genes were connected to the hearing in the ancestral bat node, suggesting an evolutionary enhancement of hearing capabilities and the initial development of echolocation in the common ancestor of bats. This result may indicate that bat ancestor has already evolved primitive echolocation, and two LE lineages evolve the echolocation independently. Our study provides new insight on the molecular underpinnings of echolocation adaptations and evolution in bats and supplies valuable resources for future studies on the transcriptomics of bat echolocation.

The mouse serves as a critical model species with an extensive bioinformatics database for exploring molecular mechanisms. The inner ear controls hearing and balance, while the temporal molecular signatures and transcriptional regulatory dynamics underlying its development are still unclear. To establish complete figures, this thesis secondly investigated time-course transcriptome in the mouse inner ear from embryonic day 11.5 (E11.5) to postnatal day 7 (P7) using bulk RNA-Seq. A total of 10,822 differentially expressed genes (DEGs) were identified between pairwise stages. We identified nine significant temporal expression profiles using time-series expression analysis. The constantly down-regulated profiles throughout the development are related to DNA activity and neurosensory development, while the constantly upregulated profiles are related to collagen and extracellular matrix. Further coexpression network analysis revealed that several hub genes, such as Pnoc, Cd9, and Krt27, are related to the neurosensory development, cell adhesion, and keratinization. We uncovered three important transcription regulatory paths during mice inner ear development. Transcription factors related to Hippo/TGFβ signaling induced decreased expressions of genes related to the neurosensory and inner ear development, while a series of INF genes activated the expressions of genes in immunoregulation. This study deepening our understanding of the temporal and regulatory mechanisms of inner ear development, and this transcriptomic data could fuel future multi-species comparative studies and elucidate the evolutionary trajectory of auditory development.

Lastly, to understand the developmental dynamics and molecular mechanism of bat high frequency hearing, we utilised RNA-Seq data to conduct comparative inner ear transcriptomics to assess the variation of gene expression among bats with three types of echolocations: constant-frequency (CF) bats Rhinolophus cornutus, frequency-modulated (FM) bats Vespertilio sinensis and non-LE bats Cynopterus sphinx. Using time-series expression analysis in CF and FM bat, we identified six significant temporal expression profiles respectively, when constantly down-regulated profiles throughout the development are related to inner ear development, while the constantly upregulated profiles are related to collagen and extracellular matrix. Coexpression analysis for FM bat identified 15 modules, and the biggest module Turquoise containing 3855 genes were enriched in inner ear related GO term. The hub gene Sox3 in module turquoise has proved that played unique roles in development of hair cells and inner ear development in mouse and zebrafish. Next, we conducted stage-by-stage cross-species comparison between CF and FM bats. In CF bats, upregulated genes were primarily involved in actin filaments and cell adhesion. In FM bats, upregulated genes were associated with the neuronal system, microtubules, and ion channels. Thirdly, we performed different expression analysis between FM and non-LE bat, and CF and non-LE bat. The upregulate gene set in FM and CF bats were enriched in neuron system and collagen and extracellular matrix development respectively, and upregulate gene sets in non-LE bat were enriched in microtubule term. Finally, Principal Component Analysis and correlation heatmap suggested that non-LE bat was closed to mouse, and two LE bats were separated to two sides. This result may indicate that non-LE bats may keep in the ancestor status of echolocation ability with mouse, and two LE lineages evolve the echolocation independently.

In summary, this thesis provides a comprehensive comparative analysis of the genomes and inner ear transcriptomes, deeply exploring the molecular mechanisms of the bat auditory system. It also offers new evidence for investigating the evolutionary history of bat LE. These findings not only contribute to solving evolutionary questions but also provide valuable molecular data for future solutions to hearing loss. Looking forward, further studies on bat inner ear should consider utilizing single-cell and spatial transcriptomics. These approaches can enhance our understanding of developmental trajectories and provide detailed maps of individual organs, thereby improving our grasp of complex developmental biology story.

Date of Award	28 Aug 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Jun LI (Supervisor), Natasza KURPIOS (External Co-Supervisor) & Daisuke KOYABU (External Co-Supervisor)