Development and regionalization of the telencephalon and peripheral associated systems in the shark Scyliorhinus canicula

This thesis represents the first monographic study of the telencephalon and peripheral systems associated to it in an elasmobranch species, Scyliorhinus canicula, which is a crucial model from an evolutionary point of view. We present a thoughtful characterization of these structures from early embryonic development to adulthood, which intend to fill a gap in the knowledge about vertebrate telencephalon evolution and, particularly, about its development. We describe for the first time the development of the peripheral olfactory system and terminal nerve system in S. canicula and present evidences of their shared origin in the migratory mass. We also demonstrate that Pax6 cells located along the developing olfactory nerve are migrating neurons that might be acting as guidepost cells for axons travelling towards the olfactory bulb. Additionally, we delimit main pallial and subpallial subdivisions by means of the analysis of expression of key developmental genes as Dlx2, Nkx2.1, Tbr1, Lhx9, Emx1, Shh and Otp. This genetic approach also allowed us to analyze different tangential migratory routes originated in the subpallium. Finally, we study the connections of the proposed basal ganglia in juveniles of S. canicula which draw interesting conclusions about the identity of the striatum and the conservation of the dopaminergic nigrostriatal pathway in elasmobranchs

Tissue-specific actions of Pax6 on the balance of proliferation and differentiation in developing forebrain are Foxg1-dependent

Summary: Differences in the growth and maturation of diverse forebrain tissues depend on region-specific transcriptional regulation. Individual transcription factors act simultaneously in multiple regions that develop very differently, raising questions about the extent to which their actions vary regionally. We found that the transcription factor Pax6 affects the transcriptomes and the balance between proliferation and differentiation in opposite directions in the diencephalon versus cerebral cortex. We tested several possible mechanisms to explain Pax6's tissue-specific actions and found that the presence of the transcription factor Foxg1 in the cortex but not in the diencephalon was most influential. We found that Foxg1 is responsible for many of the differences in cell cycle gene expression between the diencephalon and cortex and, in cortex lacking Foxg1, Pax6's action on the balance of proliferation versus differentiation becomes diencephalon like. Our findings reveal a mechanism for generating regional forebrain diversity in which one transcription factor completely reverses the actions of another. : Neuroscience; Molecular Neuroscience; Developmental Neuroscience Subject Areas: Neuroscience, Molecular Neuroscience, Developmental Neuroscienc

Regionalization of the Shark Hindbrain: A Survey of an Ancestral Organization

Cartilaginous fishes (chondrichthyans) represent an ancient radiation of vertebrates currently considered the sister group of the group of gnathostomes with a bony skeleton that gave rise to land vertebrates. This out-group position makes chondrichthyans essential in assessing the ancestral organization of the brain of jawed vertebrates. To gain knowledge about hindbrain evolution we have studied its development in a shark, the lesser spotted dogfish Scyliorhinus canicula by analyzing the expression of some developmental genes and the origin and distribution of specific neuronal populations, which may help to identify hindbrain subdivisions and boundaries and the topology of specific cell groups. We have characterized three developmental periods that will serve as a framework to compare the development of different neuronal systems and may represent a suitable tool for comparing the absolute chronology of development among vertebrates. The expression patterns of Pax6, Wnt8, and HoxA2 genes in early embryos of S. canicula showed close correspondence to what has been described in other vertebrates and helped to identify the anterior rhombomeres. Also in these early embryos, the combination of Pax6 with protein markers of migrating neuroblasts (DCX) and early differentiating neurons (general: HuC/D; neuron type specific: GAD, the GABA synthesizing enzyme) revealed the organization of S. canicula hindbrain in both transverse segmental units corresponding to visible rhombomeres and longitudinal columns. Later in development, when the interrhombomeric boundaries fade away, accurate information about S. canicula hindbrain subdivisions was achieved by comparing the expression patterns of Pax6 and GAD, serotonin (serotoninergic neurons), tyrosine hydroxylase (catecholaminergic neurons), choline acetyltransferase (cholinergic neurons), and calretinin (a calcium-binding protein). The patterns observed revealed many topological correspondences with other vertebrates and led to reconsideration of the current view of the elasmobranch hindbrain segmentation as peculiar among vertebrates

Identifying foetal forebrain interneurons as a target for monogenic autism risk factors and the polygenic 16p11.2 microdeletion

BACKGROUND: Autism spectrum condition or 'autism' is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2 +/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. METHODS: We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2 +/- rat cerebral cortex and hippocampus at post-natal day (P) 21. RESULTS: We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2 +/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. LIMITATIONS: The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2 +/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. CONCLUSIONS: We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2 +/- rats. </p

The Shark Alar Hypothalamus:Molecular Characterization of Prosomeric Subdivisions and Evolutionary Trends

The hypothalamus is an important physiologic center of the vertebrate brain involved in the elaboration of individual and species survival responses. To better understand the ancestral organization of the alar hypothalamus we revisit previous data on ScOtp, ScDlx2/5, ScTbr1, ScNkx2.1 expression and Pax6 immunoreactivity jointly with new data on ScNeurog2, ScLhx9, ScLhx5, and ScNkx2.8 expression, in addition to immunoreactivity to serotonin (5-HT) and doublecourtin (DCX) in the catshark Scyliorhinus canicula, a key species for this purpose since cartilaginous fishes are basal representatives of gnathostomes (jawed vertebrates). Our study revealed a complex genoarchitecture for the chondrichthyan alar hypothalamus. We identified terminal (rostral) and peduncular (caudal) subdivisions in the prosomeric paraventricular and subparaventricular areas (TPa/PPa and TSPa/PSPa, respectively) evidenced by the expression pattern of developmental genes like ScLhx5 (TPa) and immunoreactivity against Pax6 (PSPa) and 5-HT (PPa and PSPa). Dorso-ventral subdivisions were only evidenced in the SPa (SPaD, SPaV; respectively) by means of Pax6 and ScNkx2.8 (respectively). Interestingly, ScNkx2.8 expression overlap over the alar-basal boundary, as Nkx2.2 does in other vertebrates. Our results reveal evidences for the existence of different groups of tangentially migrated cells expressing ScOtp, Pax6 and ScDlx2. The genoarchitectonic comparative analysis suggests alternative interpretations of the rostral-most alar plate in prosomeric terms and reveals a conserved molecular background for the vertebrate alar hypothalamus likely acquired before/during the agnathan-gnathostome transition, on which Otp, Pax6, Lhx5, and Neurog2 are expressed in the Pa while Dlx and Nkx2.2/Nkx2.8 are expressed in the SPa

Identifying foetal forebrain interneurons as a target for monogenic autism risk factors and the polygenic 16p11.2 microdeletion

Abstract Background Autism spectrum condition or ‘autism’ is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2 +/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. Methods We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8–26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2 +/- rat cerebral cortex and hippocampus at post-natal day (P) 21. Results We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2 +/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. Limitations The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2 +/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. Conclusions We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2 +/- rats