Adhesion molecule Amigo2 is involved in the fasciculation process of the fasciculus retroflexus.

BACKGROUND: The fasciculus retroflexus is the prominent efferent pathway from the habenular complex. Medial habenular axons form a core packet whereas lateral habenular axons course in a surrounding shell. Both groups of fibers share the same initial pathway but differ in the final segment of the tract, supposedly regulated by surface molecules. The gene Amigo2 codes for a membrane adhesion molecule with an immunoglobulin-like domain 2 and is selectively expressed in the medial habenula. We present it as a candidate for controlling the fasciculation behavior of medial habenula axons. RESULTS: First, we studied the development of the habenular efferents in an Amigo2 lack of function mouse model. The fasciculus retroflexus showed a variable defasciculation phenotype. Gain of function experiments allowed us to generate a more condensed tract and rescued the Amigo2 knock-out phenotype. Changes in Amigo2 function did not alter the course of habenular fibers. CONCLUSION: We have demonstrated that Amigo2 plays a subtle role in the fasciculation of the fasciculus retroflexus.

Temporal Dynamics and Neuronal Specificity of Grin3a Expression in the Mouse Forebrain.

GluN3A subunits endow N-Methyl-D-Aspartate receptors (NMDARs) with unique biophysical, trafficking, and signaling properties. GluN3A-NMDARs are typically expressed during postnatal development, when they are thought to gate the refinement of neural circuits by inhibiting synapse maturation, and stabilization. Recent work suggests that GluN3A also operates in adult brains to control a variety of behaviors, yet a full spatiotemporal characterization of GluN3A expression is lacking. Here, we conducted a systematic analysis of Grin3a (gene encoding mouse GluN3A) mRNA expression in the mouse brain by combining high-sensitivity colorimetric and fluorescence in situ hybridization with labeling for neuronal subtypes. We find that, while Grin3a mRNA expression peaks postnatally, significant levels are retained into adulthood in specific brain regions such as the amygdala, medial habenula, association cortices, and high-order thalamic nuclei. The time-course of emergence and down-regulation of Grin3a expression varies across brain region, cortical layer of residence, and sensory modality, in a pattern that correlates with previously reported hierarchical gradients of brain maturation and functional specialization. Grin3a is expressed in both excitatory and inhibitory neurons, with strong mRNA levels being a distinguishing feature of somatostatin interneurons. Our study provides a comprehensive map of Grin3a distribution across the murine lifespan and paves the way for dissecting the diverse functions of GluN3A in health and disease.

Molecular regionalization of the developing amphioxus neural tube challenges major partitions of the vertebrate brain.

All vertebrate brains develop following a common Bauplan defined by anteroposterior (AP) and dorsoventral (DV) subdivisions, characterized by largely conserved differential expression of gene markers. However, it is still unclear how this Bauplan originated during evolution. We studied the relative expression of 48 genes with key roles in vertebrate neural patterning in a representative amphioxus embryonic stage. Unlike nonchordates, amphioxus develops its central nervous system (CNS) from a neural plate that is homologous to that of vertebrates, allowing direct topological comparisons. The resulting genoarchitectonic model revealed that the amphioxus incipient neural tube is unexpectedly complex, consisting of several AP and DV molecular partitions. Strikingly, comparison with vertebrates indicates that the vertebrate thalamus, pretectum, and midbrain domains jointly correspond to a single amphioxus region, which we termed Di-Mesencephalic primordium (DiMes). This suggests that these domains have a common developmental and evolutionary origin, as supported by functional experiments manipulating secondary organizers in zebrafish and mice.

Growth and differentiation factor 10 (Gdf10) is involved in Bergmann glial cell development under Shh regulation.

Growth differentiation factor 10 (Gdf10), also known as Bmp3b, is a member of the transforming growth factor (TGF)-ß superfamily. Gdf10 is expressed in Bergmann glial cells, which was investigated by single-cell transcriptional profiling (Koirala and Corfas, (2010) PLoS ONE 5: e9198). Here we provide a detailed characterization of Gdf10 expression from E14, the stage at which Gdf10 is expressed for the first time in the cerebellum, until P28. We detected Gdf10 expression in both germinal zones: in the ventricular zone (VZ) of the 4th ventricle as well as in the rhombic lip (RL). The VZ has been postulated to give rise to GABAergic neurons and glial cells, whereas the RL gives rise to glutamatergic neurons. Thus, it was very surprising to discover a gene that is expressed exclusively in glial cells and is not restricted to an expression in the VZ, but is also present in the RL. At postnatal stages Gdf10 was distributed equally in Bergmann glial cells of the cerebellum. Furthermore, we found Gdf10 to be regulated by Sonic hedgehog (Shh), which is secreted by Purkinje cells of the cerebellum. In the conditional Shh mutants, glial cells showed a reduced expression of Gdf10, whereas the expression of Nestin and Vimentin was unchanged. Thus, we show for the first time, that Gdf10, expressed in Bergmann glial cells, is affected by the loss of Shh as early as E18.5, suggesting a regulation of glial development by Shh.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

Stria medullaris innervation follows the transcriptomic division of the habenula.

The habenula is a complex neuronal population integrated in a pivotal functional position into the vertebrate limbic system. Its main afference is the stria medullaris and its main efference the fasciculus retroflexus. This neuronal complex is composed by two main components, the medial and lateral habenula. Transcriptomic and single cell RNAseq studies have unveiled the morphological complexity of both components. The aim of our work was to analyze the relation between the origin of the axonal fibers and their final distribution in the habenula. We analyzed 754 tracing experiments from Mouse Brain Connectivity Atlas, Allen Brain Map databases, and selected 12 neuronal populations projecting into the habenular territory. Our analysis demonstrated that the projections into the medial habenula discriminate between the different subnuclei and are generally originated in the septal territory. The innervation of the lateral habenula displayed instead a less restricted distribution from preoptic, terminal hypothalamic and peduncular nuclei. Only the lateral oval subnucleus of the lateral habenula presented a specific innervation from the dorsal entopeduncular nucleus. Our results unveiled the necessity of novel sorts of behavioral experiments to dissect the different functions associated with the habenular complex and their correlation with the distinct neuronal populations that generate them.

Gli2-Mediated Shh Signaling Is Required for Thalamocortical Projection Guidance.

The thalamocortical projections are part of the most important higher level processing connections in the vertebrates and follow a highly ordered pathway from their origin in the thalamus to the cerebral cortex. Their functional complexities are not only due to an extremely elaborate axon guidance process but also due to activity-dependent mechanisms. Gli2 is an intermediary transcription factor in the Sonic hedgehog (Shh) pathway. During neural early development, Shh has an important role in dorsoventral patterning, diencephalic anteroposterior patterning, and many later developmental processes, such as axon guidance and cell migration. Using a Gli2 knockout mouse line, we have studied the role of Shh signaling mediated by Gli2 in the development of the thalamocortical projections during embryonic development. In wild-type brains, we have described the normal trajectory of the thalamocortical axons into the context of the prosomeric model. Then, we have compared it with the altered thalamocortical axons course in Gli2 homozygous embryos. The thalamocortical axons followed different trajectories and were misdirected to other territories probably due to alterations in the Robo/Slit signaling mechanism. In conclusion, the alteration of Gli2-mediated Shh signaling produces an erroneous specification of several territories related with the thalamocortical axons. This is translated into a huge modification in the pathfinding signaling mechanisms needed for the correct wiring of the thalamocortical axons.

Cerebellar oligodendroglial cells have a mesencephalic origin.

While the origin of oligodendroglia in the prosencephalon and spinal cord has been extensively studied and accurately described, the origin of this cell type in the cerebellum is largely unknown. To investigate where cerebellar oligodendrocytes generate and which migratory pathways they follow to reach their final destination in the adult, in ovo transplants were performed using the quail/chick chimeric system. The chimeric embryos were developed up to HH43-49 (17-19 days of incubation) to map the location of donor cells and analyze their phenotype by immunohistochemistry. As a result, mesencephalic homotopic and homochronic transplants generated cellular migratory streams moving from the grafted epithelium into the host cerebellum, crossing the isthmus mainly through the velum medullare and invading the central white matter. From here, these mesencephalic cells invaded all the layers of the cerebellar cortex except the granular layer. The majority of the cells were detected in the central and folial white matter, as well as in superficial regions of the internal granular layer, surrounding the Purkinje cells. In the latter case, the donor cells presented a Bergmann glial morphology and were Vimentin positive, while in other areas they were PLP and Olig2-positive, indicating an oligodendroglial fate. The combinatory analysis of the different grafts allowed us to propose the fate map of chick cerebellar oligodendroglia at the neural tube stage. As a result, the majority of the cerebellar oligodendrocytes originate from the parabasal plate of the mesencephalon.

Dynamic expression patterns of Nkx6.1 and Nkx6.2 in the developing mes-diencephalic basal plate.

The components of the molecular codes needed to specify the different neuronal populations present in the basal neural tube are being identified. These codes become more intricate as we move to more anterior regions of the central nervous system. The aim of this study is to thoroughly analyze the expression pattern of Nkx6.1, Nkx6.2, and Pou4f1. These three genes are candidates to play an important role in the determination and differentiation of the basal nuclei of the mesencephalon and diencephalon. The results obtained have shown that there is a longitudinal domain positive for both Nkx6.1 and Nkx6.2 that is medial to the Pou4f1-positive red nucleus. This domain could correspond to part of the reticular formation, which extends from the diencephalon and the mesencephalon. The nuclei integrated in this domain would be the rostral interstitial nucleus, the interstitial nucleus of Cajal, and a mesencephalic equivalent to these nuclei.

Tangential Intrahypothalamic Migration of the Mouse Ventral Premamillary Nucleus and Fgf8 Signaling.

The tuberal hypothalamic ventral premamillary nucleus (VPM) described in mammals links olfactory and metabolic cues with mating behavior and is involved in the onset of puberty. We offer here descriptive and experimental evidence on a migratory phase in the development of this structure in mice at E12.5-E13.5. Its cells originate at the retromamillary area (RM) and then migrate tangentially rostralward, eschewing the mamillary body, and crossing the molecularly distinct perimamillary band, until they reach a definitive relatively superficial ventral tuberal location. Corroborating recent transcriptomic studies reporting a variety of adult glutamatergic cell types in the VPM, and different projections in the adult, we found that part of this population heterogeneity emerges already early in development, during tangential migration, in the form of differential gene expression properties of at least 2-3 mixed populations possibly derived from subtly different parts of the RM. These partly distribute differentially in the core and shell parts of the final VPM. Since there is a neighboring acroterminal source of Fgf8, and Fgfr2 is expressed at the early RM, we evaluated a possible influence of Fgf8 signal on VPM development using hypomorphic Fgf8neo/null embryos. These results suggested a trophic role of Fgf8 on RM and all cells migrating tangentially out of this area (VPM and the subthalamic nucleus), leading in hypomorphs to reduced cellularity after E15.5 without alteration of the migrations proper.

Wnt1 Role in the Development of the Habenula and the Fasciculus Retroflexus.

Wnt1 is one of the morphogenes that controls the specification and differentiation of neuronal populations in the developing central nervous system. The habenula is a diencephalic neuronal complex located in the most dorsal aspect of the thalamic prosomere. This diencephalic neuronal population is involved in the limbic system and its malfunction is related with several psychiatric disorders. Our aim is to elucidate the Wnt1 role in the habenula and its main efferent tract, the fasciculus retroflexus, development. In order to achieve these objectives, we analyzed these structures development in a Wnt1 lack of function mouse model. The habenula was generated in our model, but it presented an enlarged volume. This alteration was due to an increment in habenular neuroblasts proliferation rate. The fasciculus retroflexus also presented a wider and disorganized distribution and a disturbed final trajectory toward its target. The mid-hindbrain territories that the tract must cross were miss-differentiated in our model. The specification of the habenula is Wnt1 independent. Nevertheless, it controls its precursors proliferation rate. Wnt1 expressed in the isthmic organizer is vital to induce the midbrain and rostral hindbrain territories. The alteration of these areas is responsible for the fasciculus retroflexus axons misroute.

Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons.

The fasciculus retroflexus is an important fascicle that mediates reward-related behaviors and is associated with different psychiatric diseases. It is the main habenular efference and constitutes a link between forebrain regions, the midbrain, and the rostral hindbrain. The proper functional organization of habenular circuitry requires complex molecular programs to control the wiring of the habenula during development. However, the mechanisms guiding the habenular axons toward their targets remain mostly unknown. Here, we demonstrate the role of the mesodiencephalic dopaminergic neurons (substantia nigra pars compacta and ventral tegmental area) as an intermediate target for the correct medial habenular axons navigation along the anteroposterior axis. These neuronal populations are distributed along the anteroposterior trajectory of these axons in the mesodiencephalic basal plate. Using in vitro and in vivo experiments, we determined that this navigation is the result of netrin 1 attraction generated by the mesodiencephalic dopaminergic neurons. This attraction is mediated by the receptor deleted in colorectal cancer (DCC), which is strongly expressed in the medial habenular axons. The increment in our knowledge on the fasciculus retroflexus trajectory guidance mechanisms opens the possibility of analyzing if its alteration in mental health patients could account for some of their symptoms.

Neuronal tangential migration from Nkx2.1-positive hypothalamus.

During the development of the central nervous system, the immature neurons suffer different migration processes. It is well known that Nkx2.1-positive ventricular layer give rise to critical tangential migrations into different regions of the developing forebrain. Our aim was to study this phenomenon in the hypothalamic region. With this purpose, we used a transgenic mouse line that expresses the tdTomato reporter driven by the promotor of Nkx2.1. Analysing the Nkx2.1-positive derivatives at E18.5, we found neural contributions to the prethalamic region, mainly in the zona incerta and in the mes-diencephalic tegmental region. We studied the developing hypothalamus along the embryonic period. From E10.5 we detected that the Nkx2.1 expression domain was narrower than the reporter distribution. Therefore, the Nkx2.1 expression fades in a great number of the early-born neurons from the Nkx2.1-positive territory. At the most caudal positive part, we detected a thin stream of positive neurons migrating caudally into the mes-diencephalic tegmental region using time-lapse experiments on open neural tube explants. Late in development, we found a second migratory stream into the prethalamic territory. All these tangentially migrated neurons developed a gabaergic phenotype. In summary, we have described the contribution of interneurons from the Nkx2.1-positive hypothalamic territory into two different rostrocaudal territories: the mes-diencephalic reticular formation through a caudal tangential migration and the prethalamic zona incerta complex through a dorsocaudal tangential migration.

Gestational Exposure to Sodium Valproate Disrupts Fasciculation of the Mesotelencephalic Dopaminergic Tract, With a Selective Reduction of Dopaminergic Output From the Ventral Tegmental Area.

Gestational exposure to valproic acid (VPA) is known to cause behavioral deficits of sociability, matching similar alterations in human autism spectrum disorder (ASD). Available data are scarce on the neuromorphological changes in VPA-exposed animals. Here, we focused on alterations of the dopaminergic system, which is implicated in motivation and reward, with relevance to social cohesion. Whole brains from 7-day-old mice born to mothers given a single injection of VPA (400 mg/kg b.wt.) on E13.5 were immunostained against tyrosine hydroxylase (TH). They were scanned using the iDISCO method with a laser light-sheet microscope, and the reconstructed images were analyzed in 3D for quantitative morphometry. A marked reduction of mesotelencephalic (MT) axonal fascicles together with a widening of the MT tract were observed in VPA treated mice, while other major brain tracts appeared anatomically intact. We also found a reduction in the abundance of dopaminergic ventral tegmental (VTA) neurons, accompanied by diminished tissue level of DA in ventrobasal telencephalic regions (including the nucleus accumbens (NAc), olfactory tubercle, BST, substantia innominata). Such a reduction of DA was not observed in the non-limbic caudate-putamen. Conversely, the abundance of TH+ cells in the substantia nigra (SN) was increased, presumably due to a compensatory mechanism or to an altered distribution of TH+ neurons occupying the SN and the VTA. The findings suggest that defasciculation of the MT tract and neuronal loss in VTA, followed by diminished dopaminergic input to the ventrobasal telencephalon at a critical time point of embryonic development (E13-E14) may hinder the patterning of certain brain centers underlying decision making and sociability.

The role of Otx genes in progenitor domains of ventral midbrain.

The mesencephalic dopaminergic (mesDA) neurons play a relevant role in the control of movement, behaviour and cognition. Indeed loss and/or abnormal development of mesDA neurons is responsible for Parkinson's disease as well as for addictive and psychiatric disorders. A wealth of information has been provided on gene functions involved in the molecular mechanism controlling identity, fate and survival of mesDA neurons. Collectively, these studies are contributing to a growing knowledge of the genetic networks required for proper mesDA development, thus disclosing new perspectives for therapeutic approaches of mesDA disorders. Here we will focus on the control exerted by Otx genes in early decisions regulating the differentiation of progenitors located in the ventral midbrain. In this context, the regulatory network involving Otx functional interactions with signalling molecules and transcription factors required to promote or prevent the development of mesDA neurons will be analyzed in detail.

The Amniote Oculomotor Complex.

The oculomotor (OM) complex is a combination of somatic and parasympatethic neurons. The correct development and wiring of this cranial pair is essential to perform basic functions: eyeball and eyelid movements, pupillary constriction, and lens accommodation. The improper formation or function of this nucleus leads pathologies such as strabismus. We describe the OM organization and function in different vertebrate brains, including chick, mouse, and human. The morphological localization is detailed, as well as the spatial relation with the trochlear nucleus in order to adjust some misleading anatomical topographic descriptions. We detailed the signaling processes needed for the specification of the OM neurons. The transcriptional programs driven the specification and differentiation of these neurons are partially determined. We summarized recent genetic studies that have led to the identification of guidance mechanisms involved in the migration, axon pathfinding, and targeting of the OM neurons. Finally, we overviewed the pathology associated to genetic malformations in the OM development and related clinical alterations. Anat Rec, 302:446-451, 2019. © 2018 Wiley Periodicals, Inc.

Overview of the History of the Cranial Nerves: From Galen to the 21st Century.

Throughout history the description and classification of the cranial nerves has been linked to the development and characteristics of anatomy and the role that it played as a tool in providing rationality to medicine, together with social, cultural, religious, and philosophical factors. Anatomists were interested in the cranial nerves, but they disagreed on their number and their paths. We can divide the history of the cranial nerves into three different periods: the first, early or macroscopic period; the second or microscopic period; and the third period or ontogenesis and genoarchitecture. The main aim of this article is to show how the description and knowledge of the cranial nerves were developed in the course of these three periods, and to highlight the main changes produced and the factors related to these changes. We describe how the first period was mainly focused on establishing the definition, number and paths of the cranial nerves, through contributions ranging from Galen's studies in the second century to Sömmerring's Doctoral Dissertation in 1778 that described 12 cranial nerves for the first time. Then, the microscopic period was concentrated on the identification of the real nuclei of origin of the different cranial nerves located in the brain stem. Finally came the third period, or ontogenesis and genoarchitecture of the rhombecephalic and mesencephalic cranial nerve nuclei. Anat Rec, 302:381-393, 2019. © 2018 Wiley Periodicals, Inc.

The Otx family.

Otx1 and Otx2, the murine homologs of the Drosophila orthodenticle gene, play a remarkable role in specification and regionalization of forebrain and midbrain. Recently, genetic approaches have indicated that OTD, OTX1 and OTX2 have retained reciprocal functional equivalence in evolution, whereas their regulatory control has been remarkably modified. This suggests that during the evolution of the vertebrate brain, regulatory changes modulating the transcriptional and translational control of pre-existing gene functions might have favored the establishment of new morphogenetic pathways.

Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain.

Organizing centers emit signaling molecules that specify different neuronal cell types at precise positions along the anterior-posterior (A-P) and dorsal-ventral (D-V) axes of neural tube during development. Here we report that reduction in Otx proteins near the alar-basal plate boundary (ABB) of murine midbrain resulted in a dorsal shift of Shh expression, and reduction in Otx proteins at the midbrain-hindbrain boundary (MHB) resulted in an anterior expansion of the Fgf8 domain. Our data thus indicate that an Otx dose-dependent repressive effect coordinates proper positioning of Shh and Fgf8 expression. Furthermore, this control is effective for conferring proper cell identity in the floor-plate region of midbrain and does not require an Otx2-specific property. We propose that this mechanism may provide both A-P and D-V positional information to neuronal precursors located within the midbrain.

Otx2 controls identity and fate of glutamatergic progenitors of the thalamus by repressing GABAergic differentiation.

GABAergic and glutamatergic neurons modulate inhibitory and excitatory networks in the CNS, and their impairment may cause neurological and psychiatric disorders. Thus, understanding the molecular mechanisms that control neurotransmitter phenotype and identity of excitatory and inhibitory progenitors has considerable relevance. Here we investigated the consequence of Otx2 (orthodenticle homolog) ablation in glutamatergic progenitors of the dorsal thalamus (referred to as thalamus). We report that Otx2 is cell-autonomously required in these progenitors to repress GABAergic differentiation. Our data indicate that Otx2 may prevent GABAergic fate switch by repressing the basic helix-loop-helix gene Mash1 (mammalian achaete-schute homolog) in progenitors expressing Ngn2 (neurogenin homolog). The lack of Otx2 also resulted in the activation of Pax3 (paired box gene), Pax7, and Lim1 (Lin-11/Isl-1/Mec-3), three genes normally coexpressed with Mash1 and GABAergic markers in the pretectum, thus suggesting that thalamic progenitors lacking Otx2 exhibit marker similarities with those of the pretectum. Furthermore, Otx2 ablation gave rise to a marked increase in proliferating activity of thalamic progenitors and the formation of hyperplastic cell masses. Thus, this study provides evidence for a novel and crucial role of Otx2 in the molecular mechanism by which identity and fate of glutamatergic precursors are established in the thalamus. Our data also support the concept that proper assignment of identity and fate of neuronal precursors occurs through the suppression of alternative differentiation programs.