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

Developmental study of the cerebellum in cartilaginous fishes: Towards the identification of primitive features of the cerebellar formation in gnathostomes

This Thesis corresponds to a developmental study of the cerebellum in the shark Scyliorhinus canicula, which belongs to the gnathostome group with most primitive features of the cerebellum. The genoarchitecture of rostral hindbrain in this species appears very similar to that of other gnathostomes, and differences from that of agnatha showed possible genetic changes at the base of the evolutionary origin of the cerebellum. The combined analysis of cerebellar morphogenesis and genoarchitecture allowed: identifying consistent anatomical landmarks highly conserved, discerning the possible basal median-lateral cerebellar compartmentalization, and providing some evidence of the exclusive cerebellar origin from rhombomere 1. The developmental study and neurochemical characterization of the precerebellar nuclei showed shared traits between S. canicula and other jawed vertebrates. Besides, some evidence that migrating pathways of precerebellar nuclei precursors appeared very early in gnathostome evolution was observed. Therefore, present results may reveal the ancestral organization of the cerebellar system

Wnt transport mechanisms during vertebrate tissue patterning

Wnt signalling is one of the key pathways regulating numerous important processes during development and adult tissue maintenance. Wnt proteins act as morphogens originating from a Wnt source forming a gradient in the responding tissues to allow pattern formation. The exact mechanism how Wnt proteins are distributed to form gradients is still poorly understood.During my thesis, I analysed in detail how Wnts are distributed to form a gradient

Rx plays multiple roles in eye development

The homeobox was first identified as a common feature of Drosophila genes that control pattern formation during embryogenesis. These genes have been implicated in the regional specialization of the developing brain, spinal cord and other body structures. Homeobox gene families are highly conserved and encode DNA-binding regulatory proteins. Certain members of the homeobox gene family, including Pax6, Chx10, Lhx2, Otx2, Six3 and Six6, are crucial for development of the eye. A new family of retinal homeobox genes, Rx, is expressed early in embryogenesis in retinal stem cells. This expression pattern, along with results of overexpression and gene deletion studies, is consistent with a role for Rx in retinal stem cell specification and proliferation. In this dissertation, studies of the Rx expression in adult organisms reveal that Rx is abundantly expressed in human and mouse adult neural retina. A number of patients with anophthalmia and microphthalmia were screened and it was found that these conditions are associated with mutations in the Rx gene. The data generated in the first two projects raise a question about a role of Rx at different stages of eye development. To address this problem, a conditional allele of Rx gene was generated. This allele retains normal Rx activity but is a subject to inactivation by Cre recombinase. Using Cre recombinase under a control of forebrain-specific promoter (Foxg1 promoter), Rx conditional inactivation is achieved. Conditional animals (Rx-Foxg1-Cre) lack eyes and optic nerves but otherwise appear to be perfectly normal, making them an ideal model for anophthalmia as seen in the patient population. Analysis of Rx-Foxg1-Cre mice indicate that Rx is critical not only for the initial steps of eye development but also for axial patterning, since both, dorso-ventral and proximo-distal markers are affected

Magnetic resonance imaging, in situ hybridization, and immunohistochemistry-based analyses of early prenatal ethanol exposure-induced central nervous system abnormalities

Fetal alcohol spectrum disorders (FASD), the collection of defects resulting from prenatal alcohol (ethanol) exposure, has been the subject of basic and clinical investigation for four decades, but remains a major public health problem. At the severe end of the spectrum is fetal alcohol syndrome (FAS), which is characterized by the presence of growth retardation, craniofacial anomalies, and brain deficits. The research described herein was designed to advance our knowledge regarding ethanol's insult to the developing brain, with much of it directed toward testing the hypothesis that the application of magnetic resonance-based imaging to the examination of brain morphology, regional volumes and fiber tracts in ethanol-exposed fetal mice would facilitate new discoveries. As with other teratogens, it is well known that the type and severity of abnormality induced by ethanol is dependent upon the dose, timing, and pattern of maternal exposure. For this study, the CNS dysmorphology resulting from acute gestational day (GD) 7 maternal ethanol administration was examined in fetal mice utilizing state of the art imaging techniques. This time in mouse development is consistent with that in the third week of human gestation. Magnetic resonance microscopy (MRM) allowed for linear, volumetric and 3-dimensional morphologic analyses of ethanol-induced alterations in the fetal CNS and diffusion tensor imaging (DTI) provided for assessment of fiber tract abnormalities. In addition, routine histological techniques were utilized for detailed examination of the ventromedian forebrain in ethanol-exposed embryos and fetuses. Major new findings from these studies include the following regarding the consequences of acute GD7 ethanol exposure in mice 1) cerebral cortical heterotopias are induced; a discovery that was facilitated by MRM-based analyses, 2) fiber tract abnormalities involving the corpus callosum, anterior commissure, and fornix/fimbria occur, as evidenced by DTI, 3) fiber tract abnormalities, as identified in fetal mice, persist into periadolescent stages, 4) ventral forebrain insult preferentially involving the preoptic area and medial ganglionic eminences reduces Olig2 and GABA expression and alters the morphology of somatostatin-expressing cells. Overall, the results of this work promise to aid in clinical recognition, diagnosis, and prevention of FASD

Molecular genetic characterization of the mouse truncate (tc) mutation

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Fate-mapping neural stem cells in the mouse ventral neural tube by Cre-lox transgenesis

Neurons and glia (astrocytes and oligodendrocytes) are the two major cell types that make up the central nervous system (CNS). They are generated from precursor domains within the neuroepithelial germinal zone (ventricular zone, VZ) that surrounds the ventricles of the brain and the central canal of the spinal cord (the embryonic neural tube). In general, neurons are generated before glia. The intra-spinal circuits that control movement and locomotion are made up of different neuronal and glial elements that develop separately but come together to form interconnected functional units. To understand the logic of circuit development and ultimately circuit-driven behaviour, it is necessary to understand where and when each type of cell originates. To identify the products of the most ventral progenitor domain in the developing spinal cord, known as (Nkx2.2-expressing p3 domain), I made use of Cre-loxP technology. I generated a transgenic mouse line that expresses an inducible form of Cre recombinase (CreERT2) under Nkx2.2 transcriptional control and crossed this with a Cre-dependant reporter mouse to visualize p3-derived progeny. I confirmed that the p3 domain generates Sim1-expressing V3 interneurons, serotonergic interneurons as well as visceral motor neurons of the hindbrain. p3 progenitors also produce two spatially restricted subtypes of astrocytes, a few oligodendrocytes and ventrallypositioned ependymal cells. Unexpectedly, my studies also revealed that pre-ganglionic motor neurons of the sympathetic nervous system (SPNs, visceral motor neurons of the thoracic spinal cord), as well as a population of dorsally-located Sim1-expressing interneurons, are produced from Nkx2.2-expressing precursors. SPNs have been generally believed to originate from the same progenitor pool as HB9-positive somatic motor neurons (sMNs), defined by expression of Olig2 (pMN domain, immediately dorsal to p3). Supporting this idea, no spinal sMNs or SPNs are formed in Olig2-null mice. However, I found that Nkx2.2-expressing p3 precursors do not generate any HB9-positive sMNs, implying that sMNs and SPNs derive from distinct precursors - the latter from the most ventral part of the pMN domain that transiently co-expresses Nkx2.2 and Olig2. Thus, segregation of SPNs and sMNs occurs already in the neuroepithelium before their post-mitotic progenitors migrate away from the VZ into the ventral horns. This is how visceral and somatic MNs are known to develop in the brainstem, so my results provide a unifying theme to MN development at different levels of the neuraxis