Molecular diversity of diencephalic astrocytes reveals adult astrogenesis regulated by Smad4.

Astrocytes regulate brain-wide functions and also show region-specific differences, but little is known about how general and region-specific functions are aligned at the single-cell level. To explore this, we isolated adult mouse diencephalic astrocytes by ACSA-2-mediated magnetic-activated cell sorting (MACS). Single-cell RNA-seq revealed 7 gene expression clusters of astrocytes, with 4 forming a supercluster. Within the supercluster, cells differed by gene expression related to ion homeostasis or metabolism, with the former sharing gene expression with other regions and the latter being restricted to specific regions. All clusters showed expression of proliferation-related genes, and proliferation of diencephalic astrocytes was confirmed by immunostaining. Clonal analysis demonstrated low level of astrogenesis in the adult diencephalon, but not in cerebral cortex grey matter. This led to the identification of Smad4 as a key regulator of diencephalic astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocytes show diverse gene expression states related to distinct functions with some subsets being more widespread while others are more regionally restricted. However, all share low-level proliferation revealing the novel concept of adult astrogenesis in the diencephalon.

Transcriptome profiling reveals expression signatures of cranial neural crest cells arising from different axial levels.

BACKGROUND: Cranial neural crest cells (NCCs) are a unique embryonic cell type which give rise to a diverse array of derivatives extending from neurons and glia through to bone and cartilage. Depending on their point of origin along the antero-posterior axis cranial NCCs are rapidly sorted into distinct migratory streams that give rise to axial specific structures. These migratory streams mirror the underlying segmentation of the brain with NCCs exiting the diencephalon and midbrain following distinct paths compared to those exiting the hindbrain rhombomeres (r). The genetic landscape of cranial NCCs arising at different axial levels remains unknown. RESULTS: Here we have used RNA sequencing to uncover the transcriptional profiles of mouse cranial NCCs arising at different axial levels. Whole transcriptome analysis identified over 120 transcripts differentially expressed between NCCs arising anterior to r3 (referred to as r1-r2 migratory stream for simplicity) and the r4 migratory stream. Eight of the genes differentially expressed between these populations were validated by RT-PCR with 2 being further validated by in situ hybridisation. We also explored the expression of the Neuropilins (Nrp1 and Nrp2) and their co-receptors and show that the A-type Plexins are differentially expressed in different cranial NCC streams. CONCLUSIONS: Our analyses identify a large number of genes differentially regulated between cranial NCCs arising at different axial levels. This data provides a comprehensive description of the genetic landscape driving diversity of distinct cranial NCC streams and provides novel insight into the regulatory networks controlling the formation of specific skeletal elements and the mechanisms promoting migration along different paths.

The conserved dopaminergic diencephalospinal tract mediates vertebrate locomotor development in zebrafish larvae.

The most conserved part of the vertebrate dopaminergic system is the orthopedia (otp)-expressing diencephalic neuronal population that constitutes the dopaminergic diencephalospinal tract (DDT). Although studies in the neonatal murine spinal cord in vitro suggest an early locomotor role of the DDT, the function of the DDT in developing vertebrates in vivo remains unknown. Here, we investigated the role of the DDT in the locomotor development of zebrafish larvae. To assess the development of the behavioral and neural locomotor pattern, we used high-throughput video tracking in combination with peripheral nerve recordings. We found a behavioral and neural correspondence in the developmental switch from an immature to mature locomotor pattern. Blocking endogenous dopamine receptor 4 (D(4)R) signaling in vivo either before or after the developmental switch prevented or reversed the switch, respectively. Spinal transections of post-switch larvae reestablished the immature locomotor pattern, which was rescued to a mature-like pattern via spinal D(4)R agonism. Selective chemogenetic ablation of otp b (otpb) neurons that contribute to the DDT perpetuated the immature locomotor pattern in vivo. This phenotype was recapitulated by diencephalic transections that removed the dopaminergic otpb population and was rescued to a mature-like locomotor pattern by D(4)R agonism. We conclude that the dopaminergic otpb population, via the DDT, is responsible for spinal D(4)R signaling to mediate the developmental switch to the mature locomotor pattern of zebrafish. These results, integrated with the mammalian literature, suggest that the DDT represents an evolutionarily conserved neuromodulatory system that is necessary for normal vertebrate locomotor development.

The ADHD-susceptibility gene lphn3.1 modulates dopaminergic neuron formation and locomotor activity during zebrafish development.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, increased impulsivity and emotion dysregulation. Linkage analysis followed by fine-mapping identified variation in the gene coding for Latrophilin 3 (LPHN3), a putative adhesion-G protein-coupled receptor, as a risk factor for ADHD. In order to validate the link between LPHN3 and ADHD, and to understand the function of LPHN3 in the etiology of the disease, we examined its ortholog lphn3.1 during zebrafish development. Loss of lphn3.1 function causes a reduction and misplacement of dopamine-positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. The behavioral phenotype can be rescued by the ADHD treatment drugs methylphenidate and atomoxetine. Together, our results implicate decreased Lphn3 activity in eliciting ADHD-like behavior, and demonstrate its correlated contribution to the development of the brain dopaminergic circuitry.

Impaired dopaminergic neuron development and locomotor function in zebrafish with loss of pink1 function.

Mutations in the human PTEN-induced kinase 1 (PINK1) gene are linked to recessive familial Parkinson's disease. Animal models of altered PINK1 function vary greatly in their phenotypic characteristics. Drosophila pink1 mutants exhibit mild dopaminergic neuron degeneration and locomotion defects. Such defects are not observed in mice with targeted null mutations in pink1, although these mice exhibit impaired dopamine release and synaptic plasticity. Here, we report that in zebrafish, morpholino-mediated knockdown of pink1 function did not cause large alterations in the number of dopaminergic neurons in the ventral diencephalon. However, the patterning of these neurons and their projections are perturbed. This is accompanied by locomotor dysfunction, notably impaired response to tactile stimuli and reduced swimming behaviour. All these defects can be rescued by expression of an exogenous pink1 that is not a target of the morpholinos used. These results indicate that normal PINK1 function during development is necessary for the proper positioning of populations of dopaminergic neurons and for the establishment of neuronal circuits in which they are implicated.

Longitudinal developmental analysis of prethalamic eminence derivatives in the chick by mapping of Tbr1 in situ expression.

The prethalamic eminence (PThE) is the most dorsal subdomain of the prethalamus, which corresponds to prosomere 3 (p3) in the prosomeric model for vertebrate forebrain development. In mammalian and avian embryos, the PThE can be delimited from other prethalamic areas by its lack of Dlx gene expression, as well as by its expression of glutamatergic-related genes such as Pax6, Tbr2 and Tbr1. Several studies in mouse embryos postulate the PThE as a source of migratory neurons that populate given telencephalic centers. Concerning the avian PThE, it is visible at early embryonic stages as a compact primordium, but its morphology becomes cryptic at perinatal stages, so that its developmental course and fate are largely unknown. In this report, we characterize in detail the ontogeny of the chicken PThE from 5 to 15 days of development, according to morphological criteria, and using Tbr1 as a molecular marker for this structure and its migratory cells. We show that initially the PThE contacts rostrally the medial pallium, the pallial amygdala and the paraventricular hypothalamic alar domain. Approximately from embryonic day 6 onwards, the PThE becomes progressively reduced in size and cell content due to massive tangential migration of many of its neuronal derivatives towards nearby subpallial and hypothalamic regions. Our analysis supports that these migratory neurons from the avian PThE target telencephalic centers such as the commissural septal nuclei, as previously described in mammals, but also the diagonal band and preoptic areas, and hypothalamic structures in the paraventricular hypothalamic area.

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway.

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.

Nuclear localization of histamine in neonatal rat brain.

The concentration of histamine in the brains of neonatal rats is considerably higher than that in adults. Subcellular fractionation studies revealed that about 90 percent of the histamine content of neonatal rat brain is confined to the crude nuclear fraction obtained by differential fractionation. Purified nuclei prepared from these fractions retained 90 percent of their histamine content. The nuclear localization of histamine in the brains of neonatal rats suggests a function for histamine in modulating the growth processes of the neonatal brain.

The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity.

The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.

Neurogenic development of the auditory areas of the midbrain and diencephalon in the Xenopus laevis and evolutionary implications.

To study whether the core-versus-shell pattern of neurogenesis occurred in the mesencephalic and diencephalic auditory areas of amniotes also appears in the amphibian, [(3)H]-thymidine was injected into tadpoles at serial developmental stages of Xenopus laevis. Towards the end of metamorphism, [(3)H]-thymidine labeling was examined and led to two main observations: 1) neuron generation in the principal nucleus (Tp) started at stage 50, and peaked at stage 53, whereas it began at stage 48.5, and peaked around stage 49 in the other two mesencephalic auditory areas, the laminar nucleus (Tl) and the magnocellular nucleus (Tmc). 2) Neuron generation appeared at stage 40, and peaked around stage 52 in the posterior thalamic nucleus (P) and the central thalamic nucleus (C). Our study revealed that, like the cores of mesencephalic auditory nuclei in amniotes, Tp showed differences from Tl and Tmc in the onset and the peak of neurogenesis. However, such differences did not occur in the P and C. Our neurogenetic data were consistent with anatomical and physiological reports indicating a clear distinction between the mesencephalic, but not the diencephalic auditory areas of the amphibian. Our data are helpful to get insights into the organization of auditory nuclei and its evolution in vertebrates.

Efferent retinal projections visualized by immunohistochemical detection of the estrogen-related receptor beta in the postnatal and adult mouse brain.

Recently, a new nuclear receptor subfamily has been identified and referred to as estrogen-related receptors. This new group shares sequence similarity, target genes, co-regulatory proteins, and action sites with the estrogen receptors; however, natural estrogens are not estrogen-related receptors ligands. One of the receptors belonging to this group, estrogen-related receptor beta (ERRbeta), is essential for embryo development and is believed to be involved in estrogen-regulated pathways. In this study, we analyzed the presence of the ERRbeta protein in the mouse brain by means of immunohistochemistry, using a commercial polyclonal antibody against ERRbeta (Sigma, E0156). This study represents the first description dealing with the immunolocalization of ERRbeta in a mammalian brain. Our results revealed numerous ERRbeta immunoreactive fibers in the retinal efferent projections in the brain, which was in agreement with the presence of intense ERRbeta immunoreactivity in the cell bodies and axonal processes of the retinal ganglion cells. In both postnatal and adult brains, ERRbeta immunoreactive fibers were distributed in a pattern which perfectly matched the retinal efferent projections: optic tract, supraoptic commissure, hypothalamic suprachiasmatic nucleus, ventral and dorsal geniculate nuclei, pretectal nuclei, and superior colliculus. Due to reliable, fine, and complete staining of the retinal axons obtained with the anti-ERRbeta antibody (E0156), we suggest that this antibody could be used as a valuable tool for labeling the full retinofugal projections in postnatal or adult brains.

Development of the diencephalic relay structures of the visual thalamofugal system in pigeons.

To compare the developmental pattern of the visual tecto- and thalamofugal pathways in the altricial pigeon, we examined the posthatch differentiation of the retinothalamic system. Choleratoxin was injected into the left and right eye to visualize the retinal innervation pattern of the lateral geniculate nucleus of the thalamus (GLd). The calcium-binding proteins parvalbumin and calbindin and GABA(Abeta) receptors were used as indicators for the functional development of the GLd. Although all retinorecipient thalamic target structures were invaded by retinal fibers directly after hatching, density of the projection increased during the first week. While the adult GLd was characterized by a substantial number of cells displaying calbindin-immunoreactivity and by a sparse innervation by parvalbumin-immunoreactive fibers, after hatching no labelling for calcium-binding proteins could be detected. Calbindin-immunoreactivity appeared not before posthatching day 7, while parvalbumin-immunoreactive fibers were detected only after the third week. In contrast, a dense but diffuse GABA(Abeta) receptor-labelling was present from hatching onwards that decreased during development. The delayed expression of calbindin as well as changes in the density of GABA(Abeta) receptors indicate that maturation of GLd neurons extends long into the posthatch period. It is likely that the GABAergic interneurons mainly develop within this posthatch timeframe. Combined with the delayed development of the parvalbumin-positive innervation, the developmental pattern of GLd neurons suggests that the thalamofugal networks are immature after hatching and therefore still sensitive to modulations of posthatch visual experience.

Early diencephalon development in Alligator.

Diencephalon development was investigated in a reptilian embryo, Alligator mississipiensis, beginning at a single compartment stage and continuing until internal subdivisions were present within major units. A variety of morphological techniques were used: immunocytochemistry, histochemistry, and cresyl violet staining. The diencephalon begins as a single unit. In the transverse domain, the diencephalon subsequently divides into two: the parencephalon and the synencephalon. The parencephalon then splits into the parencephalon anterior and parencephalon posterior. Still later, the synencephalon undergoes parcellation into the synencephalon anterior and synencephalon posterior. Subsequently, internal subdivisions occur in each of these four compartments. When the diencephalon has become subdivided into two compartments and continuing until internal subdivisions are present in each unit, a longitudinal border separating a dorsal, presumed alar plate, from a ventral, presumed basal plate, was seen. No clear cut subunits were reliably identified in the telencephalon or secondary prosencephalon during this period of early development in Alligator. Early diencephalon development in birds (chick) and mammals (humans) follows a similar pattern. Specifically, a single diencephalic compartment divides into two zones: the parencephalon and synencephalon. Subsequently, the parencephalon becomes subdivided into an anterior and posterior unit. Some studies, including the present one, have noted further parcellation of the synencephalon into an anterior and posterior component, whereas others have not. Notwithstanding differences as to whether the synencephalon is a single unit or not, these detailed analyses in reptiles (Alligator), birds (chick), and mammals (humans), suggest that the initial pattern of early diencephalon development in amniotes is similar.

Developmental origin and fate of meso-diencephalic dopamine neurons.

Specific vulnerability of substantia nigra compacta neurons as compared to ventral tegmental area neurons, as emphasized in Parkinson's disease, has been studied for many years and is still not well understood. The molecular codes and mechanisms that drive development of these structures have recently been studied through the use of elegant genetic ablation experiments. The data suggested that specific genes at specific anatomical positions in the ventricular zone are crucial to drive development of young neurons into the direction of the dopaminergic phenotype. In addition, it has become clear the these dopaminergic neurons are present in the diencephalon and in the mesencephalon and that they may contain a specific molecular signature that defines specific subsets in terms of position and function. The data indicate that these specific subsets may explain the specific response of these neurons to toxins and genetic ablation.

Signals from the caudal diencephalon are required for the projection of the Interstitial Nuclei of Cajal.

Axonal projection is controlled by discrete regions localized at the neuroepithelium, guiding the neurite growth during embryonic development. These regions exert their effect through the expression of a family of chemotropic molecules, which actively participate in the formation of neuronal connections of the central nervous system in vertebrates. Previous studies describe prosomere 1 (P1) as a possible organizer of axonal growth of the rostral rhombencephalon, contributing to the caudal projection of reticulospinal rhombencephalic neurons. This work studies the contribution of chemotropic signals from P1 or pretectal medial longitudinal fascicle (MLF) neurons upon the caudal projection of the interstitial nuclei of Cajal (INC). By using in ovo surgeries, retrograde axonal labeling, and immunohistochemical techniques, we were able to determine that the absence of P1 generates a failure in the INC caudal projection, while drastically diminishing the reticulospinal rhombencephalic neurons projections. The lack of INC projection significantly decreases the number of reticulospinal neurons projecting to the MLF. We found a 48.6% decrease in the projections to the MLF from the rostral and bulbar areas. Similarly, the observed decrease at prosomere 2 was 51.5%, with 61.8% and 32.4% for prosomeres 3 and 4, respectively; thus, constituting the most affected rostral regions. These results suggest the following possibilities: i, that the axons of the reticulospinal neurons employ the INC projection as a scaffold, fasciculating with this pioneer projection; and ii, that the P1 region, including pretectal MLF neurons, exerts a chemotropic effect upon the INC caudal projection. Nonetheless the identification of these chemotropic signals is still a pending task.

Full: Ontogenesis and development of the nonhuman primate pulvinar.

Recent evidence demonstrates that the pulvinar nuclei play a critical role in shaping the connectivity and function of the multiple cortical areas they connect. Surprisingly, however, little is known about the development of this area, the largest corpus of the thalamic nuclei, which go on to occupy 40% of the adult thalamus in the human. It was proposed that the nonhuman primate and the human pulvinar develop according to very different processes, with a greatly reduced neurogenic period in nonhuman primate compared to human and divergent origins. In the marmoset monkey, we demonstrate that neurons populating the pulvinar are generated throughout gestation, suggesting that this aspect of development is more similar to the human than first predicted. While we were able to confirm the diencephalic source of pulvinar neurons, we provide new evidence contesting the presence of an additional niche in the telencephalon. Finally, our study defines new molecular markers that will simplify future investigations in the development and evolution of the pulvinar.

Expression of brain-derived neurotrophic factor in the rat forebrain and upper brain stem during postnatal development: an immunohistochemical study.

The present study was undertaken to characterize the regional and temporal patterns of brain-derived neurotrophic factor (BDNF) in the rat forebrain and upper brain stem during postnatal development using an immunohistochemical approach. Results indicated that BDNF-immunoreactive (IR) cells could be divided into three groups based on their postnatal developmental patterns: (group 1) BDNF-IR cells were first detected between postnatal days (PND) 1 and 7, and thereafter they increased in number and remained stable during later stages of ontogeny; (group 2) BDNF-IR cells progressively increased in number with age, and then decreased in adults; (group 3) numerous BDNF-IR cells detected between PND 1 and 7 showed a dramatic reductions in number with few IR cells in adults. In contrast, the developmental pattern of most BDNF-IR fibers differed from that of IR neurons, i.e. they appeared between PND 1-28 and thereafter continued to increase in number showing a maximum level in adults. Additionally, BDNF-IR cells in the superficial layer of the neocortex and IR fibers in the stratum oriens of CA2 first appeared as late as PND 28 and in adults, respectively. After colchicine treatment, reexpression or a marked increase in the number of BDNF-IR neurons was observed in many areas of the adult brain where a progressive decrease in BDNF-IR cell numbers during development and scant or some IR neurons in adults were shown. These results showed both transient and persistent expression of BDNF in various regions of the developing rat brain.

Sex differences in the control of neuronal nitric oxide synthase by GABA-A receptors in the developing rat diencephalon.

The nitric oxide free radical (NO(*)), which is synthesized by neuronal nitric oxide synthase (nNOS), is known to play an important morphogenetic role in the developing rat brain. In the cortex, the levels of nNOS are regulated by phosphorylated cAMP response element binding protein (pCREB) downstream of GABA-A receptor activation. During early stages of neonatal development, binding of GABA to its type A receptors leads to depolarization of the neuronal membrane. One of the developmental processes mediated through GABA-A receptors is the sexual differentiation of the brain. In the present work, we investigated the effect of GABA-A receptor activation on nNOS and pCREB immunoreactivity in the developing diencephalon of 5-day-old male and female rats. Our results showed that in the bed nucleus of the stria terminalis activation of GABA-A receptors leads to increased numbers of nNOS, and pCREB as well as nNOS-pCREB doubly immunopositive cells only in the males while in the posterior hypothalamus this effect is observed in both sexes. The GABA-A receptor-mediated increase in nNOS and pCREB is abolished when L-type voltage-gated Ca(2+) channels are blocked. These results indicate that the following mechanism could be operating in a gonadal hormone-dependent and brain area-specific manner during neonatal rat brain development: Depolarization following GABA-A receptor activation leads to opening of L-type voltage-gated calcium channels, resulting in an increased Ca(2+) influx, which in turn leads to phosphorylation, and thus activation of the transcription factor CREB; the phosphorylated CREB can then induce nNOS.

Differential regulation of the zebrafish orthopedia 1 gene during fate determination of diencephalic neurons.

BACKGROUND: The homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development. RESULTS: Here, we identified two otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO) (anterior alar plate) and the posterior tuberculum (PT) (posterior basal plate). The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF. CONCLUSION: In this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.

Leaning to the left: laterality in the zebrafish forebrain.

How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left-right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left-right biases are established and propagated in the developing vertebrate brain.