Exogenous LRRK2G2019S induces parkinsonian-like pathology in a nonhuman primate

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease among the elderly. To understand pathogenesis and to test therapies, animal models that faithfully reproduce key pathological PD hallmarks are needed. As a prelude to developing a model of PD, we tested the tropism, efficacy, biodistribution, and transcriptional impact of canine adenovirus type 2 (CAV-2) vectors in the brain of Microcebus murinus, a nonhuman primate that naturally develops neurodegenerative lesions. We show that introducing helper-dependent (HD) CAV-2 vectors results in long-term, neuron-specific expression at the injection site and in afferent nuclei. Although HD CAV-2 vector injection induced a modest transcriptional response, no significant adaptive immune response was generated. We then generated and tested HD CAV-2 vectors expressing LRRK2 (leucine-rich repeat kinase 2) and LRRK2 carrying a G2019S mutation (LRRK2G2019S), which is linked to sporadic and familial autosomal dominant forms of PD. We show that HD-LRRK2G2019S expression induced parkinsonian-like motor symptoms and histological features in less than 4 months

Discovering novel mechanisms of human cortical development & disease using in vivo mouse model and in vitro human-derived cerebral organoids

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside

Discovering novel mechanisms of human cortical development & disease using in vivo mouse model and in vitro human-derived cerebral organoids

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside

Discovering novel mechanisms of human cortical development disease using in vivo mouse model and in vitro human-derived cerebral organoids.

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside. <br

Slow Inhibition and Inhibitory Recruitment in the Hippocampal Dentate Gyrus

L’hippocampe joue un rôle central dans la navigation spatiale, la mémoire et l’organisation spatio-temporelle des souvenirs. Ces fonctions sont maintenues par la capacité du gyrus denté (GD) de séparation des patrons d'activité neuronales. Le GD est situé à l’entrée de la formation hippocampique où il reconnaît la présence de nouveaux motifs parmi la densité de signaux afférant arrivant par la voie entorhinale (voie perforante). Le codage parcimonieux est la marque distinctive du GD. Ce type de codage est le résultat de la faible excitabilité intrinsèque des cellules granulaires (CGs) en combinaison avec une inhibition locale prédominante. En particulier, l’inhibition de type « feedforward » ou circuit inhibiteur antérograde, est engagée par la voie perforante en même temps que les CGs. Ainsi les interneurones du circuit antérograde fournissent des signaux GABAergique aux CGs de manière presque simultanée qu’elles reçoivent les signaux glutamatergiques. Cette thèse est centrée sur l’étude des interactions entre ces signaux excitateurs de la voie entorhinale et les signaux inhibiteurs provenant des interneurones résidant dans le GD et ceci dans le contexte du codage parcimonieux et le patron de décharge en rafale caractéristique des cellules granulaires. Nous avons adressé les relations entre les projections entorhinales et le réseau inhibitoire antérograde du GD en faisant des enregistrements électrophysiologiques des CG pendant que la voie perforante est stimulée de manière électrique ou optogénétique. Nous avons découvert un nouvel mécanisme d’inhibition qui apparait à délais dans les CGs suite à une stimulation dans les fréquences gamma. Ce mécanisme induit une hyperpolarisation de longue durée (HLD) et d’une amplitude prononce. Cette longue hyperpolarisation est particulièrement prolongée et dépasse la durée d’autres types d’inhibition transitoire lente décrits chez les CGs. L’induction de HLD crée une fenêtre temporaire de faible excitabilité suite à laquelle le patron de décharge des CGs et l’intégration d’autres signaux excitateurs sont altérés de manière transitoire. Nous avons donc conclu que l’activité inhibitrice antérograde joue un rôle central dans les processus de codage dans le GD. Cependant, alors qu’il existe une multitude d’études décrivant les interneurones qui font partie de ce circuit inhibiteur, la question de comment ces cellules sont recrutées par la voie entorhinale reste quelque peu explorée. Pour apprendre plus à ce sujet, nous avons enregistré des interneurones résidant iii dans la couche moléculaire du GD tout en stimulant la voie perforante de manière optogénétique. Cette méthode de stimulation nous a permis d’induire la libération de glutamate endogène des terminales entorhinales et ainsi d’observer le recrutement purement synaptique d’interneurones. De manière surprenante, les résultats de cette expérience démontrent un faible taux d’activation des interneurones, accompagné d’un tout aussi faible nombre total de potentiels d’action émis en réponse à la stimulation même à haute fréquence. Ce constat semble contre-intuitif étant donné qu’en générale on assume qu’une forte activité inhibitrice est requise pour le maintien du codage parcimonieux. Tout de même, l’analyse des patrons de décharge des interneurones qui ont été activés a fait ressortir la prééminence de trois grands types: décharge précoce, retardée ou régulière par rapport le début des pulses lumineux. Les résultats obtenus durant cette thèse mettent la lumière sur l’important conséquences fonctionnelles des interactions synaptique et polysynaptique de nature transitoire dans les réseaux neuronaux. Nous aimerions aussi souligner l’effet prononcé de l’inhibition à court terme du type prolongée sur l’excitabilité des neurones et leurs capacités d’émettre des potentiels d’action. De plus que cet effet est encore plus prononcé dans le cas de HLD dont la durée dépasse souvent la seconde et altère l’intégration d’autres signaux arrivants simultanément. Donc on croit que les effets de HLD se traduisent au niveau du réseaux neuronal du GD comme une composante cruciale pour le codage parcimonieux. En effet, ce type de codage semble être la marque distinctive de cette région étant donné que nous avons aussi observé un faible niveau d’activation chez les interneurones. Cependant, le manque d’activité accrue du réseau inhibiteur antérograde peut être compensé par le maintien d’un gradient GABAergique constant à travers le GD via l’alternance des trois modes de décharges des interneurones. En conclusion, il semble que le codage parcimonieux dans le GD peut être préservé même en absence d’activité soutenue du réseau inhibiteur antérograde et ceci grâce à des mécanismes alternatives d’inhibition prolongée à court terme.The hippocampus is implicated in spatial navigation, the generation and recall of memories, as well as their spatio-temporal organization. These functions are supported by the processes of pattern separation that occurs in the dentate gyrus (DG). Situated at the entry of the hippocampal formation, the DG is well placed to detect and sort novelty patterns amongst the high-density excitatory signals that arrive via the entorhinal cortex (EC). A hallmark of the DG is sparse encoding that is enabled by a combination of low intrinsic excitability of the principal cells and local inhibition. Feedforward inhibition (FFI) is recruited directly by the EC and simultaneously with the granule cells (GCs). Therefore, FFI provides fast GABA release and shapes input integration at the millisecond time scale. This thesis aimed to investigate the interplay of entorhinal excitatory signals with GCs and interneurons, from the FFI in the DG, in the framework of sparse encoding and GC’s characteristic burst firing. We addressed the long-range excitation – local inhibitory network interactions using electrophysiological recordings of GCs – while applying an electrical or optogenetic stimulation of the perforant path (PP) in the DG. We discovered and described a novel delayed-onset inhibitory post synaptic potential (IPSP) in GCs, following PP stimulation in the gamma frequency range. Most importantly, the IPSP was characterized by a large amplitude and prolonged decay, outlasting previously described slow inhibitory events in GCs. The long-lasting hyperpolarization (LLH) caused by the slow IPSPs generates a low excitability time window, alters the GCs firing pattern, and interferes with other stimuli that arrive simultaneously. FFI is therefore a key player in the computational processes that occurs in the DG. However, while many studies have been dedicated to the description of the various types of the interneurons from the FFI, the question of how these cells are synaptically recruited by the EC remains not entirely elucidated. We tackled this problem by recording from interneurons in the DG molecular layer during PP-specific optogenetic stimulation. Light-driven activation of the EC terminals enabled a purely synaptic recruitment of interneurons via endogenous glutamate release. We found that this method of stimulation recruits only a subset of interneurons. In addition, the total number of action potentials (AP) was surprisingly low even at high frequency stimulation. This result is counterintuitive, as strong and persistent inhibitory signals are assumed to restrict GC v activation and maintain sparseness. However, amongst the early firing interneurons, late and regular spiking patterns were clearly distinguishable. Interestingly, some interneurons expressed LLH similar to the GCs, arguing that it could be a commonly used mechanism for regulation of excitability across the hippocampal network. In summary, we show that slow inhibition can result in a prolonged hyperpolarization that significantly alters concurrent input’s integration. We believe that these interactions contribute to important computational processes such as sparse encoding. Interestingly, sparseness seems to be the hallmark of the DG, as we observed a rather low activation of the interneuron network as well. However, the alternating firing of ML-INs could compensate the lack of persistent activity by the continuous GABA release across the DG. Taken together these results offer an insight into a mechanism of feedforward inhibition serving as a sparse neural code generator in the DG

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