Cortical interneurons: fit for function and fit to function? Evidence from development and evolution

Cortical inhibitory interneurons form a broad spectrum of subtypes. This diversity suggests a division of labor, in which each cell type supports a distinct function. In the present era of optimisation-based algorithms, it is tempting to speculate that these functions were the evolutionary or developmental driving force for the spectrum of interneurons we see in the mature mammalian brain. In this study, we evaluated this hypothesis using the two most common interneuron types, parvalbumin (PV) and somatostatin (SST) expressing cells, as examples. PV and SST interneurons control the activity in the cell bodies and the apical dendrites of excitatory pyramidal cells, respectively, due to a combination of anatomical and synaptic properties. But was this compartment-specific inhibition indeed the function for which PV and SST cells originally evolved? Does the compartmental structure of pyramidal cells shape the diversification of PV and SST interneurons over development? To address these questions, we reviewed and reanalyzed publicly available data on the development and evolution of PV and SST interneurons on one hand, and pyramidal cell morphology on the other. These data speak against the idea that the compartment structure of pyramidal cells drove the diversification into PV and SST interneurons. In particular, pyramidal cells mature late, while interneurons are likely committed to a particular fate (PV vs. SST) during early development. Moreover, comparative anatomy and single cell RNA-sequencing data indicate that PV and SST cells, but not the compartment structure of pyramidal cells, existed in the last common ancestor of mammals and reptiles. Specifically, turtle and songbird SST cells also express the Elfn1 and Cbln4 genes that are thought to play a role in compartment-specific inhibition in mammals. PV and SST cells therefore evolved and developed the properties that allow them to provide compartment-specific inhibition before there was selective pressure for this function. This suggest that interneuron diversity originally resulted from a different evolutionary driving force and was only later co-opted for the compartment-specific inhibition it seems to serve in mammals today. Future experiments could further test this idea using our computational reconstruction of ancestral Elfn1 protein sequences.Peer Reviewe

From locomotor behavior to cerebellum evolution and development in squamate models

Locomotor behavior, the entire set of movements an individual utilizes to modify its spatial location in time, is a crucial attribute of an organism’s life. Though not responsible for movement initiation or rhythmic locomotor pattern generation, the cerebellum, an ancient and functionally conserved feature of the vertebrate brain, plays a key role in many aspects of motor performance. Variations in its morphology, relative size and cortical organization, likely resulting from divergent developmental programs, have been observed even in closely related vertebrate species, often reflecting a tight linkage between cerebellar organization and functional demands associated with ecologically relevant factors and distinct behavioral traits. Taking advantage of the extraordinary ecomorphological diversity of squamates (lizards and snakes) and adopting a multidisciplinary approach, this thesis explores the impact of locomotor behavior on squamate brain, particularly on different levels of cerebellar biological organization, and investigates cerebellar morphogenesis in two squamate species to gain insights on the developmental mechanisms potentially responsible for squamate cerebellar divergence. Along with significant variations in cerebellar morphology and relative size across squamates, this thesis first highlights a wide heterogeneity in Purkinje cell (PC) spatial layout as well as in gene expression pattern, all correlating with specific locomotor behaviors, unveiling unique relationships between a major evolutionary transition and organ specialization in vertebrates. At the developmental level, the thesis indicates that developmental features considered, so far, exclusive hallmarks of avian and mammalian cerebellogenesis characterize squamate cerebellar morphogenesis. Furthermore, the thesis suggests that variations in the spatiotemporal patterning of different cerebellar neurons could be, at least partially, at the base of the large phenotypic diversification of the squamate cerebellum. Finally, this thesis reveals that squamates provide an important framework to expand our knowledge on organ system-ecology relationships and central nervous system (CNS) development and evolution in vertebrates.Eliöiden toimintaan liittyy oleellisena osana niiden kyky liikkua, eli siirtyä paikasta toiseen erilaisten ruuminosien liikkeiden avulla. Pikkuaivot (cerebellum) ovat hyvin oleellinen osa selkärankaisten liikkeen säätelyä, ja niiden toiminta onkin säilynyt peruspiirteiltään samana selkärankaisten evoluution aikana. Vaikka pikkuaivojen rooliin ei kuulu liikkeen aloittaminen tai rytmisen liikkeen tahdin säätely, niillä on huomattava rooli muussa liikkeen säätelyssä. Tähän lukeutuvat esimerkiksi liikkeiden oppiminen ja korjaaminen. Pikkuaivoissa esiintyy hyvin paljon lajien välistä vaihtelua, mikä johtuu todennäköisesti yksilönkehityksen ja sen säätelyn eroavaisuuksista eri lajeilla. Eroja on havaittavissa niin pikkuaivojen morfologiassa, suhteellisessa koossa kuin myös niiden kuorikerroksen rakenteessa, usein jopa lähisukuisten lajien välillä. Nämä eroavaisuudet heijastelevatkin usein eläinten erilaisia toiminnallisia tarpeita, liittyen varsinkin käyttäytymispiirteisiin sekä muihin niiden ekologiaan linkittyviin tekijöihin. Suomumatelijoilla (liskoilla ja käärmeillä) on huomattava laaja kirjos erilaisia ekomorfologioita ja liikkumistapoja. Tämä väitöskirja keskittyykin selvittämään liikkumistapojen vaikutusta suomumatelijoiden aivoihin sekä yleisesti että erityisesti pikkuaivoja tarkastellen. Huomio keskittyy pikkuaivoissa sekä kokonaiskuvan muodostamiseen niiden rakenteesta että niiden yksilönkehitykseen. Yksilönkehityksen suhteen vertailussa ovat kaksi eri suomumatelijoiden edustajaa mahdollisten yksilönkehityksen muutosten mekanismien selvittämiseksi. Väitöskirjatyössä havaittiin suomumatelijoilla merkittävää pikkuaivojen morfologian ja suhteellisen koon lajienvälistä vaihtelua. Tämän lisäksi työn aikana havaittiin huomattavia eroja pikkuaivojen niin sanottujen Purkinjen solujen järjestäytymisessä sekä eri geenien luennassa erilaista liikkumistyyppiä edustavien lajien välillä. Purkinjen solujen järjestäytymisen ja geeniluennan havaittiin myös korreloivan erilaisten liikkumistyyppien kanssa, tuoden esiin mielenkiintoisen yhteyden evolutiivisten muutosten ja elinten erikoistumisen välillä. Samoin tulokset viittaavat siihen, että linnuille ja nisäkkäille ainutlaatuisiksi luultuja pikkuaivojen muodostumisen piirteitä löytyy myös suomumatelijoilta. Väitöskirjatyössä havaittiin lisäksi viitteitä suomumatelijoiden pikkuaivojen monimuotoisuuden taustalla olevista yksilönkehityksen muutoksista. Tulosten valossa on mahdollista, että pikkuaivojen neuronien kaavoituksen ajoituksen ja sijainnin muutokset voisivat ainakin osin olla syy suomumatelijoiden pikkuaivojen monimuotoisuuteen. Laajemmassa mielessä tulokset tuovat esiin myös suomumatelijoiden erittäin oleellisen roolin selkärankaisten evoluution tutkimuksessa kahdesta oleellisesta tulokulmasta: selkärankaisten keskushermoston yksilönkehityksen ja evoluution tutkimus sekä yleisemmällä tasolla elinsysteemien ja ekologian yhteyden selvittäminen

Cortical interneurons: fit for function and fit to function? Evidence from development and evolution

Cortical inhibitory interneurons form a broad spectrum of subtypes. This diversity suggests a division of labor, in which each cell type supports a distinct function. In the present era of optimisation-based algorithms, it is tempting to speculate that these functions were the evolutionary or developmental driving force for the spectrum of interneurons we see in the mature mammalian brain. In this study, we evaluated this hypothesis using the two most common interneuron types, parvalbumin (PV) and somatostatin (SST) expressing cells, as examples. PV and SST interneurons control the activity in the cell bodies and the apical dendrites of excitatory pyramidal cells, respectively, due to a combination of anatomical and synaptic properties. But was this compartment-specific inhibition indeed the function for which PV and SST cells originally evolved? Does the compartmental structure of pyramidal cells shape the diversification of PV and SST interneurons over development? To address these questions, we reviewed and reanalyzed publicly available data on the development and evolution of PV and SST interneurons on one hand, and pyramidal cell morphology on the other. These data speak against the idea that the compartment structure of pyramidal cells drove the diversification into PV and SST interneurons. In particular, pyramidal cells mature late, while interneurons are likely committed to a particular fate (PV vs. SST) during early development. Moreover, comparative anatomy and single cell RNA-sequencing data indicate that PV and SST cells, but not the compartment structure of pyramidal cells, existed in the last common ancestor of mammals and reptiles. Specifically, turtle and songbird SST cells also express the Elfn1 and Cbln4 genes that are thought to play a role in compartment-specific inhibition in mammals. PV and SST cells therefore evolved and developed the properties that allow them to provide compartment-specific inhibition before there was selective pressure for this function. This suggest that interneuron diversity originally resulted from a different evolutionary driving force and was only later co-opted for the compartment-specific inhibition it seems to serve in mammals today. Future experiments could further test this idea using our computational reconstruction of ancestral Elfn1 protein sequences

Dendritic spikes control synaptic plasticity and somatic output in cerebellar Purkinje cells.

Neurons receive the vast majority of their input onto their dendrites. Dendrites express a plethora of voltage-gated channels. Regenerative, local events in dendrites and their role in the information transformation in single neurons are, however, poorly understood. This thesis investigates the basic properties and functional roles of dendritic spikes in cerebellar Purkinje cells using whole-cell patch clamp recordings from the dendrites and soma of rat Purkinje cells in brain slices. I show that parallel fibre (PF) evoked dendritic spikes are mediated by calcium channels, depend on membrane potential and stimulus intensity and are highly localized to the spiny branches receiving the synaptic input. A determining factor in the localization and spread of dendritic calcium spikes is the activation of large-conductance, calcium dependent potassium (BK) channels. I provide a strong link between dendritic spikes and the endocannabinoid dependent short-term synaptic plasticity, depolarization-induced suppression of excitation (DSE). Gating the dendritic spikes using stimulus intensity or membrane potential, I show that the threshold of DSE is identical to that of the dendritic spikes and the extent of DSE depends on the number of dendritic spikes. Blocking BK channels increases the spatial spread of dendritic spikes and enables current injection or climbing fibre (CF) evoked dendritic spikes to suppress PF inputs via DSE. By monitoring dendritic spikes during strong PF stimulation-induced long-term depression (LTD), I also provide a link between long-term synaptic plasticity and dendritic excitability. By showing that blocking CB1 cannabinoid receptors reduces the intensity requirement for LTD, I provide a connection between the short- and long-term changes in PF strength triggered by dendritic spikes I also investigate the effect dendritic spikes have on somatic action potential output. Contrary to pyramidal cells, where dendritic spikes boost the output of the neuron, the average Purkinje cell output becomes independent from the output strength for inputs triggering dendritic spikes. However, the temporal pattern of the output is strongly affected by dendritic spikes. I show that this phenomenon depends on BK channel activation resulting in a pause in somatic firing following dendritic spikes. In summary, I present a description of PF evoked local dendritic spikes and demonstrate their functional role in controlling the synaptic input and action potential output of cerebellar Purkinje cells

Development and Evolution of the Mammalian Cerebellum at Single Cell Resolution

Originally thought to only take part in motor control, the cerebellum emerged over the last decades as an important organ in various higher cognitive functions, such as learning and speech. Besides this, the cerebellum is associated to various diseases, such as spinocerebellar ataxia, autism spectrum disorder, and medulloblastoma. The basic structure and connective properties of it are well understood, but single-cell-technologies made it possible to study the cerebellum at higher resolution. Many questions about molecular details of its development and evolution are still not answered. Cerebella are present in all jawed vertebrates, though structural diversity is macroscopic and microscopic detectable, such as the number of deep nuclei, the presence of the vermis, or the mode of production of one of the most important cell types in the cerebellum - granule cells. Using single-nucleus RNA-sequencing (snRNA-seq) and bioinformatic approaches, I studied cerebellum data of human, mouse (Mus musculus) and opossum (Monodelphis domestica). The dataset contained samples spanning the organs development at high temporal resolution. It was possible to track the differentiation of the major cerebellar neuronal and glial cell types, as well as identify states and subtypes. This generated a comprehensive map of cellular complexity through eutherian (human and mouse) and marsupial (opossum) development. Leveraging the evolutionary distance of approximately 160 million years between the eutherian and marsupial lineage, conserved and diverged cell type marker genes could be identified which might be promising candidates for understanding the basic blueprint of cerebellar cell type identity. Stage correspondence mapping aligned the vastly different developmental time frames of the three studied species and allowed the identification of a two-fold increase in Purkinje cell progenitors in the human lineage, which might be connected to a recently identified human-specific secondary ventricular zone progenitor pool. It was possible to model the differentiation path of granule and Purkinje cells from early progenitors to mature neurons. Conserved and diverged gene expression trajectories were discovered. Using in vitro and in vivo intollerance scores, I could show that genes which are dynamically expressed during differentiation show higher functional constraint as non-dynamic genes, fitting to previous bulk-RNA-seq studies, showing similar results across the development of the full organ. Some orthologs with diverging patterns were disease-associated genes, which could have implications on clinical research on conditions like autism spectrum disorders and medulloblastoma. Furthermore, fundamental changes of gene expressions, established as gain or loss of expression within a cell type and species, were detected. Affected genes showed decreased functional constraint, verifying evolutionary principles on single cell scale. Taken together, this study shows the strength of state of the art methodology combined with high resolution developmental sampling in an evolution biological context to discover fundamental principles of organ development at single-cell scale

The role of Tenascin-R in human neurodevelopmental disorders associated with cerebellar dysfunctions

Environ 500 000 enfants au Canada souffrent de maladies génétiques rares. Chacune de ces pathologies causant divers problèmes de santé et touchant un nombre restreint d'individus, nos connaissances des mécanismes sous-jacents et des possibles approches thérapeutiques sont ainsi limitées. Néanmoins, les progrès actuels des technologies de séquençage de l'ADN permettent désormais de découvrir efficacement de nouveaux gènes impliqués dans les maladies neuronales. Grâce à cette approche, le gène de la Tenascin R (TNR) a récemment été identifié comme étant à l'origine d'une maladie neurologique rare. Jusqu'ici, il a été montré chez un enfant souffrant de troubles du développement neurologique que des mutations de la TNR sont associées à une ataxie cérébelleuse et un retard de développement global. TNR est une glycoprotéine de la matrice extracellulaire exclusivement exprimée dans le système nerveux central. Elle participe à la régulation de l'extension et la régénération de l'axone, mais également à la synaptogenèse, la croissance et la migration neuronales. Néanmoins, nos connaissances du rôle de la TNR dans les processus neurodéveloppementaux se basent sur des travaux réalisés chez des rongeurs, et la fonction de cette protéine au cours du développement du cerveau humain demeure inconnue. L'objectif de mon projet de recherche est d'investiguer le profil développemental de cellules progénitrices neuronales humaines (NPCs) issues du patient mentionné ci-dessus, et de déterminer si les anomalies observées au sein du cerveau humain présentant une mutation de TNR sont liées à une altération de la migration, maturation ou encore intégration fonctionnelle des neurones. Grâce à ces travaux, il sera possible d'acquérir des informations importantes sur la fonction de la TNR dans la migration et la maturation des neurones humains. Ce programme de recherche approfondira également notre compréhension des mécanismes fondamentaux régulant le développement neuronal des NPCs issues de patients, ceci étant essentiel à la conception de stratégies thérapeutiques ainsi qu'à la validation de médicaments.Approximately 500,000 children in Canada are affected by rare genetic disorders. Each specific disorder causes several health problems and affects a small number of individuals, therefore our knowledge about mechanisms underlying the disease and possible therapeutic interventions are strongly limited. However, the progress in DNA sequencing technologies now provides an effective way to discover new genes involved in neuronal diseases. Using this innovative approach, Tenascin R (TNR) gene has been recently identified as novel rare neurological disease-causing gene. So far, it has been showed, in a child affected by neurodevelopmental disorder, that mutations in TNR correlate with cerebellar ataxia and global development delay. TNR is a member of extracellular matrix glycoproteins and is exclusively expressed in the central nervous system. TNR contributes to the regulation of axon extension and regeneration, but also to synaptogenesis, neuronal growth and migration. However, our knowledge about the role of TNR in different neurodevelopmental processes is based on experimental work performed in rodents, and the function of this protein in human brain development remains unknown. The aim of this research project is to study the developmental profile of human neuronal progenitor cells (NPCs) derived from the above-mentioned patient and control subjects and to determine whether abnormalities observed in the human brain with TNR mutation are linked to affected neuronal migration, maturation or functional integration. This work will provide crucial information on TNR function during migration and maturation of human neurons. This research project will also deepen our understanding of fundamental mechanisms regulating neuronal development of patient-derived NPCs which will be crucial for designing treatment strategies and drug testing/validation

Generation and Characterization of the Cation-Chloride Cotransporter KCC2 Hypomorphic Mouse

The cation-Cl- cotransporter (CCC) family comprises of Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporters (NKCC1-2), and four K+-Cl- cotransporters (KCC1-4). These proteins are involved in several physiological activities, such as cell volume regulation. In neuronal tissues, NKCC1 and KCC2 are important in determining the intracellular Cl- levels and hence the neuronal responses to inhibitory neurotransmitters GABA and glycine. One aim of the work was to elucidate the roles for CCC isoforms in the control of nervous system development. KCC2 mRNA was shown to be developmentally up-regulated and follow neuronal maturation, whereas NKCC1 and KCC4 transcripts were highly expressed in the proliferative zones of subcortical regions. KCC1 and KCC3 mRNA displayed low expression throughout the embryogenesis. These expression profiles suggest a role for CCC isoforms in maturation of synaptic responses and in the regulation of neuronal proliferation during embryogenesis. The major aim of this work was to study the biological consequences of KCC2-deficiency in the adult CNS, by generating transgenic mice retaining 15-20% of normal KCC2 levels. In addition, by using these mice as a tool for in vivo pharmacological analysis, we investigated the requirements for KCC2 in tonic versus phasic GABAA receptor-mediated inhibition. KCC2-deficient mice displayed normal reproduction and life span, but showed several behavioral abnormalities, including increased anxiety-like behavior, impaired performance in water maze, alterations in nociceptive processing, and increased seizure susceptibility. In contrast, the mice displayed apparently normal spontaneous locomotor activity and motor coordination. Pharmacological analysis of KCC2-deficient mice revealed reduced sensititivity to diazepam, but normal gaboxadol-induced sedation, neurosteroid hypnosis and alcohol-induced motor impairment. Electrophysiological recordings from CA1-CA3 subregions of the hippocampus showed that KCC2 deficiency affected the reversal potentials of both the phasic and tonic GABA currents, and that the tonic conductance was not affected. The results suggest that requirement for KCC2 in GABAergic neurotransmission may differ among several functional systems in the CNS, which is possibly due to the more critical role of KCC2 activity in phasic compared to tonic GABAergic inhibition.Kationi-kloridi kuljetusproteiini-perhe koostuu natrium-kloridi (NCC), natrium-kalium-kloridi (NKCC1-2), sekä kalium-kloridi (KCC1-4) kuljetusproteiineista. Nämä proteiinit ovat osallisena useissa solujen fysiologissa prosesseissa, kuten solujen tilavuuden säätely. KCC2 ja NKCC1 ovat erityisen tärkeitä hermokudoksessa, jossa ne säätelevät solujen kloridipitoisuutta ja täten hermoston estävien välittäjäaineiden γ-aminovoihapon (GABA) ja glysiinin toimintaa. Yksi työn tavoitteista oli selventää näiden proteiinien rooleja hermoston kehityksessä. KCC2 mRNA:n ilmenemisen havaittiin voimistuvan tuntuvasti kehityksen kuluessa ja seuraavan hermoston kehitystä. NKCC1 ja KCC4 mRNA:t ilmentyivät voimakkaimmin niillä aivojen alueilla, joissa solut jakaantuvat neurogeneesin aikana. KCC1 ja KCC3 mRNA:t ilmentyivät vähäisesti koko sikiönkehityksen ajan. Tulokset puoltavat mahdollista roolia kationi-kloridi kuljetusproteiineille synapsien vasteiden kehityksessä, sekä hermosolujen jakautumisen säätelyssä. Työn päätavoitteena oli tuottaa KCC2-hypomorfinen hiiri (n. 80 % puutos) ja tutkia tämän hiiren ilmiasun muutoksia. Käytimme kyseistä hiirimallia myös selvittääksemme farmakologisesti KCC2:n osuutta GABAA-reseptorikompleksin välittämässä faasisessa ja toonisessa neurotransmissiossa. KCC2:n puutos ilmeni käyttäytymisen tasolla. Hiiri oli ahdistushäiriöinen ja selkeästi normaalia kouristusherkempi, sekä osoitti muistihäiriöitä ja normaalia korkeamman kipukynnyksen. Hiiren motorinen koordinaatio ja spontaani lokomotorinen aktiivisuus olivat sitä vastoin ilmeisen normaalilla tasolla. KCC2-hypomorfisen hiiren farmakologinen analyysi osoitti hiiren olevan epäherkkä bentsodiatsepiini diatsepaamille, mutta normaalisti herkkä gaboksadolin sedaatiovasteelle, neurosteroidin hypnoosivasteelle sekä alkoholin motoriikkaa huonontavalle vasteelle. Sähköfysiologiset mittaukset hippokampuksessa osoittivat KCC2 puutoksen vaikuttavan GABAA-välitteisten faasisten ja toonisten sähkövirtojen käänteispotentiaaleihin, mutta GABAA-välitteinen tooninen konduktanssi oli ilmeisen normaali. Nämä tulokset antavat olettaa että GABA neurotransmissiossa KCC2:n rooli voi vaihdella riippuen siitä, mitä keskushermoston eri funktionaalisista järjestelmistä aktivoidaan. Tämä voi johtua KCC2:n kriittisemmästä roolista GABAA-välitteisessä faasisessa neurotransmissiossa verrattuna tooniseen neurotransmissioon

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