Spatially resolved cell atlas of the teleost telencephalon and deep homology of the vertebrate forebrain.

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking variations in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely "everted" telencephalon, which has confounded comparisons of their brain regions to other vertebrates. Here we combine spatial transcriptomics and single nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the Mchenga conophorus cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell-types in the fish telencephalon and subpallial, hippocampal, and cortical cell-types in tetrapods, and find support for partial eversion of the teleost telencephalon. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain.

Specific biomarkers and neurons distribution of different brain regions in largemouth bass (Micropterus salmoides).

The brain regulates multiple physiological processes in fish. Despite this, knowledge about the basic structure and function of distinct brain regions in non-model fish species remains limited due to their diversity and the scarcity of common biomarkers. In the present study, four major brain parts, the telencephalon, diencephalon, mesencephalon and rhombencephalon, were isolated in largemouth bass, Micropterus salmoides. Within these parts, nine brain regions and 74 nuclei were further identified through morphological and cytoarchitectonic analysis. Transcriptome analysis revealed a total of 7153 region-highly expressed genes and 176 region-specifically expressed genes. Genes related to growth, reproduction, emotion, learning, and memory were significantly overexpressed in the olfactory bulb and telencephalon (OBT). Feeding and stress-related genes were in the hypothalamus (Hy). Visual system-related genes were predominantly enriched in the optic tectum (OT), while vision and hearing-related genes were widely expressed in the cerebellum (Ce) region. Sensory input and motor output-related genes were in the medulla oblongata (Mo). Osmoregulation, stress response, sleep/wake cycles, and reproduction-related genes were highly expressed in the remaining brain (RB). Three candidate marker genes were further identified for each brain regions, such as neuropeptide FF (npff) for OBT, pro-melanin-concentrating hormone (pmch) for Hy, vesicular inhibitory amino acid transporter (viaat) for OT, excitatory amino acid transporter 1 (eaat1) for Ce, peripherin (prph) for Mo, and isotocin neurophysin (itnp) for RB. Additionally, the distribution of seven neurotransmitter-type neurons and five types of non-neuronal cells across different brain regions were analyzed by examining the expression of their marker genes. Notably, marker genes for glutamatergic and GABAergic neurons showed the highest expression levels across all brain regions. Similarly, the marker gene for radial astrocytes exhibited high expression compared to other markers, while those for microglia were the least expressed. Overall, our results provide a comprehensive overview of the structural and functional characteristics of distinct brain regions in the largemouth bass, which offers a valuable resource for understanding the role of central nervous system in regulating physiological processes in teleost.

Cell coupling compensates for changes in single-cell Her6 dynamics and provides phenotypic robustness.

This paper investigates the effect of altering the protein expression dynamics of the bHLH transcription factor Her6 at the single-cell level in the embryonic zebrafish telencephalon. Using a homozygote endogenous Her6:Venus reporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that the fusion of protein destabilisation (PEST) domain alters its expression dynamics, causing most cells to downregulate Her6 prematurely. However, counterintuitively, oscillatory cells increase, with some expressing Her6 at high levels, resulting in increased heterogeneity of Her6 expression in the population. These tissue-level changes appear to be an emergent property of coupling between single-cells, as revealed by experimentally disrupting Notch signalling and by computationally modelling alterations in Her6 protein stability. Despite the profound differences in the single-cell Her6 dynamics, the size of the telencephalon is only transiently altered and differentiation markers do not exhibit significant differences early on; however, a small increase is observed at later developmental stages. Our study suggests that cell coupling provides a compensation strategy, whereby an almost normal phenotype is maintained even though single-cell gene expression dynamics are abnormal, granting phenotypic robustness.

Investigation of the mechanisms underlying the development and evolution of folds of the cerebrum using gyrencephalic ferrets.

The mammalian cerebrum has changed substantially during evolution, characterized by increases in neurons and glial cells and by the expansion and folding of the cerebrum. While these evolutionary alterations are thought to be crucial for acquiring higher cognitive functions, the molecular mechanisms underlying the development and evolution of the mammalian cerebrum remain only partially understood. This is, in part, because of the difficulty in analyzing these mechanisms using mice only. To overcome this limitation, genetic manipulation techniques for the cerebrum of gyrencephalic carnivore ferrets have been developed. Furthermore, successful gene knockout in the ferret cerebrum has been accomplished through the application of the CRISPR/Cas9 system. This review mainly highlights recent research conducted using gyrencephalic carnivore ferrets to investigate the mechanisms underlying the development and evolution of cortical folds.

Input and Output Connections of the Crow Nidopallium Caudolaterale.

The avian telencephalic structure nidopallium caudolaterale (NCL) functions as an analog to the mammalian prefrontal cortex. In crows, corvid songbirds, it plays a crucial role in higher cognitive and executive functions. These functions rely on the NCL's extensive telencephalic connections. However, systematic investigations into the brain-wide connectivity of the NCL in crows or other songbirds are lacking. Here, we studied its input and output connections by injecting retrograde and anterograde tracers into the carrion crow NCL. Our results, mapped onto a published carrion crow brain atlas, confirm NCL multisensory connections and extend prior pigeon findings by identifying a novel input from the hippocampal formation. Furthermore, we analyze crow NCL efferent projections to the arcopallium and report newly identified arcopallial neurons projecting bilaterally to the NCL. These findings help to clarify the role of the NCL as central executive hub in the corvid songbird brain.

Age-related reductions in whole brain mass and telencephalon volume in very old white Carneau pigeons (Columba livia).

While studies have identified age-related cognitive impairment in pigeons (Columba livia), no study has detected the brain atrophy which typically accompanies cognitive impairment in older mammals. Instead, Coppola and Bingman (Aging is associated with larger brain mass and volume in homing pigeons (Columba livia), Neurosci. Letters 698 (2019) 39-43) reported increased whole brain mass and telencephalon volume in older, compared to younger, homing pigeons. One reason for this unexpected finding might be that the older pigeons studied were not old enough to display age-related brain atrophy. Therefore, the current study repeated Coppola and Bingman, but with a sample of older white Carneau pigeons that were on average 5.34 years older. Brains from young and old homing pigeons were weighed and orthogonal measurements of the telencephalon, cerebellum, and optic tectum were obtained. Despite having a heavier body mass than younger pigeons, older pigeons had a significant reduction in whole brain mass and telencephalon volume, but not cerebellum or optic tectum volume. This study is therefore the first to find that pigeons experience age-related brain atrophy.

GPS tracking technology and re-visiting the relationship between the avian visual Wulst and homing pigeon navigation.

Within their familiar areas homing pigeons rely on familiar visual landscape features and landmarks for homing. However, the neural basis of visual landmark-based navigation has been so far investigated mainly in relation to the role of the hippocampal formation. The avian visual Wulst is the telencephalic projection field of the thalamofugal pathway that has been suggested to be involved in processing lateral visual inputs that originate from the far visual field. The Wulst is therefore a good candidate for a neural structure participating in the visual control of familiar visual landmark-based navigation. We repeatedly released and tracked Wulst-lesioned and control homing pigeons from three sites about 10-15 km from the loft. Wulst lesions did not impair the ability of the pigeons to orient homeward during the first release from each of the three sites nor to localise the loft within the home area. In addition, Wulst-lesioned pigeons displayed unimpaired route fidelity acquisition to a repeated homing path compared to the intact birds. However, compared to control birds, Wulst-lesioned pigeons displayed persistent oscillatory flight patterns across releases, diminished attention to linear (leading lines) landscape features, such as roads and wood edges, and less direct flight paths within the home area. Differences and similarities between the effects of Wulst and hippocampal lesions suggest that although the visual Wulst does not seem to play a direct role in the memory representation of a landscape-landmark map, it does seem to participate in influencing the perceptual construction of such a map.

Prosaposin maintains adult neural stem cells in a state associated with deep quiescence.

In most vertebrates, adult neural stem cells (NSCs) continuously give rise to neurons in discrete brain regions. A critical process for maintaining NSC pools over long periods of time in the adult brain is NSC quiescence, a reversible and tightly regulated state of cell-cycle arrest. Recently, lysosomes were identified to regulate the NSC quiescence-proliferation balance. However, it remains controversial whether lysosomal activity promotes NSC proliferation or quiescence, and a finer influence of lysosomal activity on NSC quiescence duration or depth remains unexplored. Using RNA sequencing and pharmacological manipulations, we show that lysosomes are necessary for NSC quiescence maintenance. In addition, we reveal that expression of psap, encoding the lysosomal regulator Prosaposin, is enriched in quiescent NSCs (qNSCs) that reside upstream in the NSC lineage and display a deep/long quiescence phase in the adult zebrafish telencephalon. We show that shRNA-mediated psap knockdown increases the proportion of activated NSCs (aNSCs) as well as NSCs that reside in shallower quiescence states (signed by ascl1a and deltaA expression). Collectively, our results identify the lysosomal protein Psap as a (direct or indirect) quiescence regulator and unfold the interplay between lysosomal function and NSC quiescence heterogeneities.

Anatomical organization of the cerebrum of the praying mantis Hierodula membranacea.

Many predatory animals, such as the praying mantis, use vision for prey detection and capture. Mantises are known in particular for their capability to estimate distances to prey by stereoscopic vision. While the initial visual processing centers have been extensively documented, we lack knowledge on the architecture of central brain regions, pivotal for sensory motor transformation and higher brain functions. To close this gap, we provide a three-dimensional (3D) reconstruction of the central brain of the Asian mantis, Hierodula membranacea. The atlas facilitates in-depth analysis of neuron ramification regions and aides in elucidating potential neuronal pathways. We integrated seven 3D-reconstructed visual interneurons into the atlas. In total, 42 distinct neuropils of the cerebrum were reconstructed based on synapsin-immunolabeled whole-mount brains. Backfills from the antenna and maxillary palps, as well as immunolabeling of γ-aminobutyric acid (GABA) and tyrosine hydroxylase (TH), further substantiate the identification and boundaries of brain areas. The composition and internal organization of the neuropils were compared to the anatomical organization of the brain of the fruit fly (Drosophila melanogaster) and the two available brain atlases of Polyneoptera-the desert locust (Schistocerca gregaria) and the Madeira cockroach (Rhyparobia maderae). This study paves the way for detailed analyses of neuronal circuitry and promotes cross-species brain comparisons. We discuss differences in brain organization between holometabolous and polyneopteran insects. Identification of ramification sites of the visual neurons integrated into the atlas supports previous claims about homologous structures in the optic lobes of flies and mantises.

Nuclei and tracts in the thalamus of crocodiles.

The thalamus is one of the most important divisions of the forebrain because it serves as the major hub for transmission of information between the brainstem and telencephalon. While many studies have investigated the thalamus in mammals, comparable analyses in reptiles are incomplete. To fill this gap in knowledge, the thalamus was investigated in crocodiles using a variety of morphological techniques. The thalamus consists of two parts: a dorsal and a ventral division. The dorsal thalamus was defined by its projections to the telencephalon, whereas the ventral thalamus lacked this circuit. The complement of nuclei in each part of the thalamus was identified and characterized. Alar and basal components of both the dorsal and ventral thalamus were distinguished. Although some alar-derived nuclei in the dorsal thalamus shared certain features, no grouping could account for all of the known nuclei. However, immunohistochemical observations suggested a subdivision of alar-derived ventral thalamic nuclei. In view of this, a different approach to the organization of the dorsal thalamus should be considered. Development of the dorsal thalamus is suggested to be one way to provide a fresh perspective on its organization.

Comparison of genes involved in brain development: insights into the organization and evolution of the telencephalic pallium.

The mechanisms underlying the organization and evolution of the telencephalic pallium are not yet clear.. To address this issue, we first performed comparative analysis of genes critical for the development of the pallium (Emx1/2 and Pax6) and subpallium (Dlx2 and Nkx1/2) among 500 vertebrate species. We found that these genes have no obvious variations in chromosomal duplication/loss, gene locus synteny or Darwinian selection. However, there is an additional fragment of approximately 20 amino acids in mammalian Emx1 and a poly-(Ala)6-7 in Emx2. Lentiviruses expressing mouse or chick Emx2 (m-Emx2 or c-Emx2 Lv) were injected into the ventricle of the chick telencephalon at embryonic Day 3 (E3), and the embryos were allowed to develop to E12-14 or to posthatchling. After transfection with m-Emx2 Lv, the cells expressing Reelin, Vimentin or GABA increased, and neurogenesis of calbindin cells changed towards the mammalian inside-out pattern in the dorsal pallium and mesopallium. In addition, a behavior test for posthatched chicks indicated that the passive avoidance ratio increased significantly. The study suggests that the acquisition of an additional fragment in mammalian Emx2 is associated with the organization and evolution of the mammalian pallium.

Telencephalic eversion in embryos and early larvae of four teleost species.

The telencephalon of ray-finned fishes undergoes eversion, which is very different to the evagination that occurs in most other vertebrates. Ventricle morphogenesis is key to build an everted telencephalon. Thus, here we use the apical marker zona occludens 1 to understand ventricle morphology, extension of the tela choroidea and the eversion process during early telencephalon development of four teleost species: giant danio (Devario aequipinnatus), blind cavefish (Astyanax mexicanus), medaka (Oryzias latipes), and paradise fish (Macroposus opercularis). In addition, by using immunohistochemistry against tubulin and calcium-binding proteins, we analyze the general morphology of the telencephalon, showing changes in the location and extension of the olfactory bulb and other telencephalic regions from 2 to 5 days of development. We also analyze the impact of abnormal eye and telencephalon morphogenesis on eversion, showing that cyclops mutants do undergo eversion despite very dramatic abnormal eye morphology. We discuss how the formation of the telencephalic ventricle in teleost fish, with its characteristic shape, is a crucial event during eversion.

Can we gain translational insights into the functional roles of cerebral cortex from acortical rodent and naturally acortical zebrafish models?

Cerebral cortex is found only in mammals and is particularly prominent and developed in humans. Various rodent models with fully or partially ablated cortex are commonly used to probe the role of cortex in brain functions and its multiple subcortical projections, including pallium, thalamus and the limbic system. Various rodent models are traditionally used to study the role of cortex in brain functions. A small teleost fish, the zebrafish (Danio rerio), has gained popularity in neuroscience research, and albeit (like other fishes) lacking cortex, its brain performs well some key functions (e.g., memory, consciousness and motivation) with complex, context-specific and well-defined behaviors. Can rodent and zebrafish models help generate insights into the role of cortex in brain functions, and dissect its cortex-specific (vs. non-cortical) functions? To address this conceptual question, here we evaluate brain functionality in intact vs. decorticated rodents and further compare it in the zebrafish, a naturally occurring acortical species. Overall, comparing cortical and acortical rodent models with naturally acortical zebrafish reveals both distinct and overlapping contributions of neocortex and 'precortical' zebrafish telencephalic regions to higher brain functions. Albeit morphologically different, mammalian neocortex and fish pallium may possess more functional similarities than it is presently recognized, calling for further integrative research utilizing both cortical and decorticated/acortical vertebrate model organisms.

Evolutionarily conserved roles of foxg1a in the developing subpallium of zebrafish embryos.

The vertebrate telencephalic lobes consist of the pallium (dorsal) and subpallium (ventral). The subpallium gives rise to the basal ganglia, encompassing the pallidum and striatum. The development of this region is believed to depend on Foxg1/Foxg1a functions in both mice and zebrafish. This study aims to elucidate the genetic regulatory network controlled by foxg1a in subpallium development using zebrafish as a model. The expression gradient of foxg1a within the developing telencephalon was examined semi-quantitatively in initial investigations. Utilizing the CRISPR/Cas9 technique, we subsequently established a foxg1a mutant line and observed the resultant phenotypes. Morphological assessment revealed that foxg1a mutants exhibit a thin telencephalon together with a misshapen preoptic area (POA). Notably, accumulation of apoptotic cells was identified in this region. In mutants at 24 h postfertilization, the expression of pallium markers expanded ventrally, while that of subpallium markers was markedly suppressed. Concurrently, the expression of fgf8a, vax2, and six3b was shifted ventrally, causing anomalous expression in regions typical of POA formation in wild-type embryos. Consequently, the foxg1a mutation led to expansion of the pallium and disrupted the subpallium and POA. This highlights a pivotal role of foxg1a in directing the dorsoventral patterning of the telencephalon, particularly in subpallium differentiation, mirroring observations in mice. Additionally, reduced expression of neural progenitor maintenance genes was detected in mutants, suggesting the necessity of foxg1a in preserving neural progenitors. Collectively, these findings underscore evolutionarily conserved functions of foxg1 in the development of the subpallium in vertebrate embryos.

Developmental loss of NMDA receptors results in supernumerary forebrain neurons through delayed maturation of transit-amplifying neuroblasts.

Developmental neurogenesis is a tightly regulated spatiotemporal process with its dysregulation implicated in neurodevelopmental disorders. NMDA receptors are glutamate-gated ion channels that are widely expressed in the early nervous system, yet their contribution to neurogenesis is poorly understood. Notably, a variety of mutations in genes encoding NMDA receptor subunits are associated with neurodevelopmental disorders. To rigorously define the role of NMDA receptors in developmental neurogenesis, we used a mutant zebrafish line (grin1-/-) that lacks all NMDA receptors yet survives to 10 days post-fertilization, offering the opportunity to study post-embryonic neurodevelopment in the absence of NMDA receptors. Focusing on the forebrain, we find that these fish have a progressive supernumerary neuron phenotype confined to the telencephalon at the end of embryonic neurogenesis, but which extends to all forebrain regions during postembryonic neurogenesis. This enhanced neuron population does not arise directly from increased numbers or mitotic activity of radial glia cells, the principal neural stem cells. Rather, it stems from a lack of timely maturation of transit-amplifying neuroblasts into post-mitotic neurons, as indicated by a decrease in expression of the ontogenetically-expressed chloride transporter, KCC2. Pharmacological blockade with MK-801 recapitulates the grin1-/- supernumerary neuron phenotype, indicating a requirement for ionotropic signaling. Thus, NMDA receptors are required for suppression of indirect, transit amplifying cell-driven neurogenesis by promoting maturational termination of mitosis. Loss of suppression results in neuronal overpopulation that can fundamentally change brain circuitry and may be a key factor in pathogenesis of neurodevelopmental disorders caused by NMDA receptor dysfunction.

A Comparison of Telencephalon Composition among Chickens, Junglefowl, and Wild Galliforms.

INTRODUCTION: Domestication is the process of modifying animals for human benefit through selective breeding in captivity. One of the traits that often diverges is the size of the brain and its constituent regions; almost all domesticated species have relatively smaller brains and brain regions than their wild ancestors. Although the effects of domestication on the brain have been investigated across a range of both mammal and bird species, almost nothing is known about the neuroanatomical effects of domestication on the world's most common bird: the chicken (Gallus gallus). METHODS: We compared the quantitative neuroanatomy of the telencephalon of white leghorn chickens with red junglefowl, their wild counterpart, and several wild galliform species. We focused specifically on the telencephalon because telencephalic regions typically exhibit the biggest differences in size in domesticate-wild comparisons. RESULTS: Relative telencephalon size was larger in chickens than in junglefowl and ruffed grouse (Bonasa umbellus). The relative size of telencephalic regions did not differ between chickens and junglefowl, but did differ in comparison with ruffed grouse. Ruffed grouse had larger hyperpallia and smaller entopallial, nidopallial, and striatal volumes than chickens and junglefowl. Multivariate analyses that included an additional three wild grouse species corroborated these findings: chicken and junglefowl have relatively larger nidopallial and striatal volumes than grouse. Conversely, the mesopallial and hyperpallial volumes tended to be relatively smaller in chickens and junglefowl. CONCLUSION: From this suite of comparisons, we conclude that chickens do not follow a pattern of widespread decreases in telencephalic region sizes that is often viewed as typical of domestication. Instead, chickens have undergone a mosaic of changes with some regions increasing and others decreasing in size, and there are few differences between chickens and junglefowl.

Conscious Experience of Stimulus Presence and Absence Is Actively Encoded by Neurons in the Crow Brain.

The emergence of consciousness from brain activity constitutes one of the great riddles in biology. It is commonly assumed that only the conscious perception of the presence of a stimulus elicits neuronal activation to signify a "neural correlate of consciousness," whereas the subjective experience of the absence of a stimulus is associated with a neuronal resting state. Here, we demonstrate that the two subjective states "stimulus present" and "stimulus absent" are represented by two specialized neuron populations in crows, corvid birds. We recorded single-neuron activity from the nidopallium caudolaterale of crows trained to report the presence or absence of images presented near the visual threshold. Because of the task design, neuronal activity tracking the conscious "present" versus "absent" percept was dissociated from that involved in planning a motor response. Distinct neuron populations signaled the subjective percepts of "present" and "absent" by increases in activation. The response selectivity of these two neuron populations was similar in strength and time course. This suggests a balanced code for subjective "presence" versus "absence" experiences, which might be beneficial when both conscious states need to be maintained active in the service of goal-directed behavior.

Involvement of nr2f genes in brain regionalization and eye development during early zebrafish development.

Nuclear receptor subfamily 2 group F (Nr2f) proteins are essential for brain development in mice, but little is known about their precise roles and their evolutionary diversification. In the present study, the expression patterns of major nr2f genes (nr2f1a, nr2f1b, and nr2f2) during early brain development were investigated in zebrafish. Comparisons of their expression patterns revealed similar but temporally and spatially distinct patterns after early somite stages in the brain. Frameshift mutations in the three nr2f genes, achieved using the CRISPR/Cas9 method, resulted in a smaller telencephalon and smaller eyes in the nr2f1a mutants; milder forms of those defects were present in the nr2f1b and nr2f2 mutants. Acridine orange staining revealed enhanced cell death in the brain and/or eyes in all nr2f homozygous mutants. The expression of regional markers in the brain did not suggest global defects in brain regionalization; however, shha expression in the preoptic area and hypothalamus, as well as fgf8a expression in the anterior telencephalon, was disturbed in nr2f1a and nr2f1b mutants, potentially leading to a defective telencephalon. Specification of the retina and optic stalk was also significantly affected. The overexpression of nr2f1b by injection of mRNA disrupted the anterior brain at a high dose, and the expression of pax6a in the eyes and fgf8a in the telencephalon at a low dose. The results of these loss- and gain-of-function approaches showed that nr2f genes regulate the development of the telencephalon and eyes in zebrafish embryos.

Different ways of evolving tool-using brains in teleosts and amniotes.

In mammals and birds, tool-using species are characterized by their relatively large telencephalon containing a higher proportion of total brain neurons compared to other species. Some teleost species in the wrasse family have evolved tool-using abilities. In this study, we compared the brains of tool-using wrasses with various teleost species. We show that in the tool-using wrasses, the telencephalon and the ventral part of the forebrain and midbrain are significantly enlarged compared to other teleost species but do not contain a larger proportion of cells. Instead, this size difference is due to large fiber tracts connecting the dorsal part of the telencephalon (pallium) to the inferior lobe, a ventral mesencephalic structure absent in amniotes. The high degree of connectivity between these structures in tool-using wrasses suggests that the inferior lobe could contribute to higher-order cognitive functions. We conclude that the evolution of non-telencephalic structures might have been key in the emergence of these cognitive functions in teleosts.

A Robust and Reproducible Method to Study Neurorepair after Stab Injury in the African Turquoise Killifish Telencephalon.

The aging population (people >60 yr old) is steadily increasing worldwide, resulting in an increased prevalence of age-related neurodegenerative diseases. Despite intensive research efforts in the past decades, there are still no therapies available to stop, cure, or prevent these diseases. Induction of successful neuroregeneration (i.e., the production of new neurons that can functionally integrate into the existing neural circuitry) could represent a therapy to replace neurons lost by injury or disease in the aged central nervous system. The African turquoise killifish, with its particularly short life span, has emerged as a useful model to study how aging influences neuroregeneration. Here, we describe a robust and reproducible stab-injury protocol to study regeneration in the telencephalon of the African turquoise killifish. After the injury, newborn cells are traced by conducting a BrdU pulse-chase experiment. To identify newborn neurons, a double immunohistochemical staining for BrdU and HuCD is carried out. Techniques such as bromodeoxyuridine (BrdU) labeling, intracardial perfusion, cryosectioning, and immunofluorescence staining are described as separate sections.