Non-coding genome contributions to the development and evolution of mammalian organs

Protein-coding sequences only cover 1-2% of a typical mammalian genome. The remaining non-coding space hides thousands of genomic elements, some of which act via their DNA sequence while others are transcribed into non-coding RNAs. Many well-characterized non-coding elements are involved in the regulation of other genes, a process essential for the emergence of different cell types and organs during development. Changes in the expression of conserved genes during development are in turn thought to facilitate evolutionary innovation in form and function. Thus, non-coding genomic elements are hypothesized to play important roles in developmental and evolutionary processes. However, challenges related to the identification and characterization of these elements, in particular in non-model organisms, has limited the study of their overall contributions to mammalian organ development and evolution. During my dissertation work, I addressed this gap by studying two major classes of non-coding elements, long non-coding RNAs (lncRNAs) and cis-regulatory elements (CREs). In the first part of my thesis, I analyzed the expression profiles of lncRNAs during the development of seven major organs in six mammals and a bird. I showed that, unlike protein-coding genes, only a small fraction of lncRNAs is expressed in reproducibly dynamic patterns during organ development. These lncRNAs are enriched for a series of features associated with functional relevance, including increased evolutionary conservation and regulatory complexity, highlighting them as candidates for further molecular characterization. I then associated these lncRNAs with specific genes and functions based on their spatiotemporal expression profiles. My analyses also revealed differences in lncRNA contributions across organs and developmental stages, identifying a developmental transition from broadly expressed and conserved lncRNAs towards an increasing number of lineage- and organ-specific lncRNAs. Following up on these global analyses, I then focused on a newly-identified lncRNA in the marsupial opossum, Female Specific on chromosome X (FSX). The broad and likely autonomous female-specific expression of FSX suggests a role in marsupial X-chromosome inactivation (XCI). I showed that FSX shares many expression and sequence features with another lncRNA, RSX — a known regulator of XCI in marsupials. Comparisons to other marsupials revealed that both RSX and FSX emerged in the common marsupial ancestor and have since been preserved in marsupial genomes, while their broad and female-specific expression has been retained for at least 76 million years of evolution. Taken together, my analyses highlighted FSX as a novel candidate for regulating marsupial XCI. In the third part of this work, I shifted my focus to CREs and their cell type-specific activities in the developing mouse cerebellum. After annotating cerebellar cell types and states based on single-cell chromatin accessibility data, I identified putative CREs and characterized their spatiotemporal activity across cell types and developmental stages. Focusing on progenitor cells, I described temporal changes in CRE activity that are shared between early germinal zones, supporting a model of cell fate induction through common developmental cues. By examining chromatin accessibility dynamics during neuronal differentiation, I revealed a gradual divergence in the regulatory programs of major cerebellar neuron types. In the final part, I explored the evolutionary histories of CREs and their potential contributions to gene expression changes between species. By comparing mouse CREs to vertebrate genomes and chromatin accessibility profiles from the marsupial opossum, I identified a temporal decrease in CRE conservation, which is shared across cerebellar cell types. However, I also found differences in constraint between cell types, with microglia having the fastest evolving CREs in the mouse cerebellum. Finally, I used deep learning models to study the regulatory grammar of cerebellar cell types in human and mouse, showing that the sequence rules determining CRE activity are conserved across mammals. I then used these models to retrace the evolutionary changes leading to divergent CRE activity between species. Collectively, my PhD work provides insights into the evolutionary dynamics of non-coding genes and regulatory elements, the processes associated with their conservation, and their contributions to the development and evolution of mammalian cell types and organs