Cortexa: a comprehensive resource for studying gene expression and alternative splicing in the murine brain.

BACKGROUND: Gene expression and alternative splicing are strictly regulated processes that shape brain development and determine the cellular identity of differentiated neural cell populations. Despite the availability of multiple valuable datasets, many functional implications, especially those related to alternative splicing, remain poorly understood. Moreover, neuroscientists working primarily experimentally often lack the bioinformatics expertise required to process alternative splicing data and produce meaningful and interpretable results. Notably, re-analyzing publicly available datasets and integrating them with in-house data can provide substantial novel insights. However, such analyses necessitate developing harmonized data handling and processing pipelines which in turn require considerable computational resources and in-depth bioinformatics expertise. RESULTS: Here, we present Cortexa-a comprehensive web portal that incorporates RNA-sequencing datasets from the mouse cerebral cortex (longitudinal or cell-specific) and the hippocampus. Cortexa facilitates understandable visualization of the expression and alternative splicing patterns of individual genes. Our platform provides SplicePCA-a tool that allows users to integrate their alternative splicing dataset and compare it to cell-specific or developmental neocortical splicing patterns. All standardized gene expression and alternative splicing datasets can be downloaded for further in-depth downstream analysis without the need for extensive preprocessing. CONCLUSIONS: Cortexa provides a robust and readily available resource for unraveling the complexity of gene expression and alternative splicing regulatory processes in the mouse brain. The data portal is available at https://cortexa-rna.com/.

Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.

Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons.

SASH1 contributes to glial cell migration in the early development of the central nervous system.

SAM and SH3 domain-containing 1 (SASH1), a member of the SLy protein family, is a tumor suppressor gene that has been studied for its association with various cancers. SASH1 is highly expressed in the mammalian central nervous system, particularly in glial cells, and is expressed in the central nervous system during zebrafish embryo development. However, SASH1's role in brain development has rarely been investigated. In this study, Morpholino oligonucleotides (MO) were used to down-regulate sash1a expression in zebrafish to observe morphological changes in the brain. Three transgenic zebrafish lines, Tg(gfap:eGFP), Tg(hb9:eGFP), and Tg(coro1a:eGFP) were selected to observe changes in glial cells, neurons, and immune cells after sash1a knockdown. Our results showed that the number of microglia residing in the developmental brain was reduced, whereas the axonal growth of caudal primary motor neurons was unaffected by sash1a downregulation. And more significantly, the gfap + glia presented abnormal arrangements and disordered orientations in sash1a morphants. The similar phenotype was verified in the mutation induced by the injection of cas9 mRNA and sash1a sgRNA. We further performed behavioral experiments in zebrafish larvae that had been injected with sash1a MO at one-cell stage, and found them exhibiting abnormal behavior trajectories. Moreover, injecting the human SASH1 mRNA rescued these phenomena in sash1a MO zebrafish. In summary, our study revealed that the downregulation of SASH1 leads to malformations in the embryonic brain and disorganization of glial cell marshalling, suggesting that SASH1 plays an important role in the migration of glial cells during embryonic brain development.

Insight into adaption to hypoxia in Tibetan chicken embryonic brains using lipidomics.

Tibetan chickens (Gallus gallus; TBCs) are a good model for studying hypoxia-related challenges. However, lipid composition in TBC embryonic brains has not been elucidated. In this study, we characterized brain lipid profiles of embryonic day 18 TBCs and dwarf laying chickens (DLCs) during hypoxia (13% O2, HTBC18, and HDLC18) and normoxia (21% O2, NTBC18, and NDLC18) by using lipidomics. A total of 50 lipid classes, including 3540 lipid molecular species, were identified and grouped into glycerophospholipids, sphingolipids, glycerolipids, sterols, prenols, and fatty acyls. Of these lipids, 67 and 97 were expressed at different levels in the NTBC18 and NDLC18, and HTBC18 and HDLC18 samples, respectively. Several lipid species, including phosphatidylethanolamines (PEs), hexosylceramides, phosphatidylcholines (PCs), and phospha-tidylserines (PSs), were highly expressed in HTBC18. These results suggest that TBCs adapt bet-ter to hypoxia than DLCs and may have distinct cell membrane composition and nervous system development, at least partly owing to differential expression of several lipid species. One tri-glyceride, one PC, one PS, and three PE lipids were identified as potential markers that discrim-inated between lipid profiles of the HTBC18 and HDLC18 samples. The present study provides valuable information about the dynamic composition of lipids in TBCs that may explain the adaptation of this species to hypoxia.

Varied hypoxia adaptation patterns of embryonic brain at different development stages between Tibetan and Dwarf laying chickens.

BACKGROUND: Tibetan chickens (Gallus gallus; TBCs), an indigenous breed distributed in the Qinghai-Tibet Plateau, are well adapted to the hypoxic environment. Currently, the molecular genetic basis of hypoxia adaptation in TBCs remains unclear. This study investigated hypoxia adaptation patterns of embryonic brain at different development stages by integrating analysis of the transcriptome with our previously published metabolome data in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed. RESULTS: During hypoxia, the results revealed that 1334, 578, and 417 differentially expressed genes (DEGs) (|log2 fold change|>1, p-value < 0.05) on days 8, 12, and 18 of development, respectively between TBCs and DLCs. Gene Ontology (GO) and pathway analyses revealed that DEGs are mainly related to metabolic pathways, vessel development, and immune response under hypoxia. This is consistent with our metabolome data that TBCs have higher energy metabolism than DLCs during hypoxia. Some vital DEGs between TBCs and DLCs, such as EPAS1, VEGFD, FBP1, FBLN5, LDHA, and IL-6 which are involved in the HIF pathway and hypoxia regulation. CONCLUSION: These results suggest varied adaptation patterns between TBCs and DLCs under hypoxia. Our study provides a basis for uncovering the molecular regulation mechanism of hypoxia adaptation in TBCs and a potential application of hypoxia adaptation research for other animals living on the Qinghai-Tibet Plateau, and may even contribute to the study of brain diseases caused by hypoxia.

Transcriptomic profiling of the developing brain revealed cell-type and brain-region specificity in a mouse model of prenatal stress.

BACKGROUND: Prenatal stress (PS) is considered as a risk factor for many mental disorders. PS-induced transcriptomic alterations may contribute to the functional dysregulation during brain development. Here, we used RNA-seq to explore changes of gene expression in the mouse fetal brain after prenatal exposure to chronic unpredictable mild stress (CUMS). RESULTS: We compared the stressed brains to the controls and identified groups of significantly differentially expressed genes (DEGs). GO analysis on up-regulated DEGs revealed enrichment for the cell cycle pathways, while down-regulated DEGs were mostly enriched in the neuronal pathways related to synaptic transmission. We further performed cell-type enrichment analysis using published scRNA-seq data from the fetal mouse brain and revealed cell-type-specificity for up- and down-regulated DEGs, respectively. The up-regulated DEGs were highly enriched in the radial glia, while down-regulated DEGs were enriched in different types of neurons. Cell deconvolution analysis further showed altered cell fractions in the stressed brain, indicating accumulation of neuroblast and impaired neurogenesis. Moreover, we also observed distinct brain-region expression pattern when mapping DEGs onto the developing Allen brain atlas. The up-regulated DEGs were primarily enriched in the dorsal forebrain regions including the cortical plate and hippocampal formation. Surprisingly, down-regulated DEGs were found excluded from the cortical region, but highly expressed on various regions in the ventral forebrain, midbrain and hindbrain. CONCLUSION: Taken together, we provided an unbiased data source for transcriptomic alterations of the whole fetal brain after chronic PS, and reported differential cell-type and brain-region vulnerability of the developing brain in response to environmental insults during the pregnancy.

Efficient gene delivery into the embryonic chicken brain using neuron-specific promoters and in ovo electroporation.

BACKGROUND: The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging. METHODS: In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters. RESULTS: Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4. CONCLUSION: We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes).

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Maternal and Adult Interleukin-17A Exposure and Autism Spectrum Disorder.

Epidemiological evidence in humans has suggested that maternal infections and maternal autoimmune diseases are involved in the pathogenesis of autism spectrum disorder. Animal studies supporting human results have shown that maternal immune activation causes brain and behavioral alterations in offspring. Several underlying mechanisms, including interleukin-17A imbalance, have been identified. Apart from the pro-inflammatory effects of interleukin-17A, there is also evidence to support the idea that it activates neuronal function and defines cognitive behavior. In this review, we examined the signaling pathways in both immunological and neurological contexts that may contribute to the improvement of autism spectrum disorder symptoms associated with maternal blocking of interleukin-17A and adult exposure to interleukin-17A. We first describe the epidemiology of maternal immune activation then focus on molecular signaling of the interleukin-17 family regarding its physiological and pathological roles in the embryonic and adult brain. In the future, it may be possible to use interleukin-17 antibodies to prevent autism spectrum disorder.

Salient brain entities labelled in P2rx7-EGFP reporter mouse embryos include the septum, roof plate glial specializations and circumventricular ependymal organs.

The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of the P2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of the P2rx7 proximal promoter, to conduct a detailed study of its distribution. We performed a comprehensive analysis of the pattern of P2X7R expression in the brain of E18.5 mouse embryos revealing interesting areas within the CNS. Particularly, strong labelling was found in the septum, as well as along the entire neural roof plate zone of the brain, except chorioidal roof areas, but including specialized circumventricular roof formations, such as the subfornical and subcommissural organs (SFO; SCO). Moreover, our results reveal what seems a novel circumventricular organ, named by us postarcuate organ (PArcO). Furthermore, this study sheds light on the ongoing debate regarding the specific presence of P2X7R in neurons and may be of interest for the elucidation of additional roles of P2X7R in the idiosyncratic histologic development of the CNS and related systemic functions.

Progenies of NG2 glia: what do we learn from transgenic mouse models ?

In the mammalian central nervous system, nerve-glia antigen 2 (NG2) glia are considered the fourth glial population in addition to astrocytes, oligodendrocytes and microglia. The fate of NG2 glia in vivo has been carefully studied in several transgenic mouse models using the Cre/loxP strategy. There is a clear agreement that NG2 glia mainly serve as progenitors for oligodendrocytes and a subpopulation of astrocytes mainly in the ventral forebrain, whereas the existence of a neurogenic potential of NG2 glia is lack of adequate evidence. This mini review summarizes the findings from recent studies regarding the fate of NG2 glia during development. We will highlight the age-and-region-dependent heterogeneity of the NG2 glia differentiation potential. We will also discuss putative reasons for inconsistent findings in various transgenic mouse lines of previous studies.

CUBIC-f: An optimized clearing method for cell tracing and evaluation of neurite density in the salamander brain.

BACKGROUND: Although tissue clearing and subsequent whole-brain imaging is now possible, standard protocols need to be adjusted to the innate properties of each specific tissue for optimal results. This work modifies exiting protocols to clear fragile brain samples and documents a downstream pipeline for image processing and data analysis. NEW METHOD: We developed a clearing protocol, CUBIC-f, which we optimized for fragile samples, such as the salamander brain. We modified hydrophilic and aqueous' tissue-clearing methods based on Advanced CUBIC by incorporating Omnipaque 350 for refractive index matching. RESULTS: By combining CUBIC-f, light sheet microscopy and bioinformatic pipelines, we quantified neuronal cell density, traced genetically marked fluorescent cells over long distance, and performed high resolution characterization of neural progenitor cells in the salamander brain. We also found that CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains. COMPARISON WITH EXITING METHODS: CUBIC-f shortens clearing and staining times, and requires less reagent use than Advanced CUBIC and Advanced CLARITY. CONCLUSION: CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains, larval and adult salamander brains which display considerable deformation using traditional CUBIC and CLARITY protocols.

Cerebral organoids as tools to identify the developmental roots of autism.

Some autism spectrum disorders (ASD) likely arise as a result of abnormalities during early embryonic development of the brain. Studying human embryonic brain development directly is challenging, mainly due to ethical and practical constraints. However, the recent development of cerebral organoids provides a powerful tool for studying both normal human embryonic brain development and, potentially, the origins of neurodevelopmental disorders including ASD. Substantial evidence now indicates that cerebral organoids can mimic normal embryonic brain development and neural cells found in organoids closely resemble their in vivo counterparts. However, with prolonged culture, significant differences begin to arise. We suggest that cerebral organoids, in their current form, are most suitable to model earlier neurodevelopmental events and processes such as neurogenesis and cortical lamination. Processes implicated in ASDs which occur at later stages of development, such as synaptogenesis and neural circuit formation, may also be modeled using organoids. The accuracy of such models will benefit from continuous improvements to protocols for organoid differentiation.

Evaluation of Fluoro-Jade C Staining: Specificity and Application to Damaged Immature Neuronal Cells in the Normal and Injured Mouse Brain.

Fluoro-Jade C (FJC) staining is widely used for the specific detection of all degenerating mature neurons, including apoptotic, necrotic, and autophagic cells. However, whether FJC staining can detect degenerating immature neurons and neural stem/precursor cells remains unclear. In addition, some conflicting studies have shown that FJC and its ancestral dyes, Fluoro-Jade (FJ) and FJB, can label resting/activated astrocytes and microglia. In the present study, we examined the validity of FJC staining for the detection of neuronal cells in adult and embryonic mouse brains under normal and injured conditions. In the adult rodent subventricular zone (SVZ)-rostral migratory stream (RMS)-olfactory bulb (OB) system, apoptosis associated with neurogenesis occurs under normal conditions. Using this system, we detected FCJ positive (+) cells, some of which were doublecortin (DCX)(+) neuroblasts, in addition to neuronal nuclei (NeuN)(+) mature neurons. FJC negative (-) apoptotic cells expressing activated Caspase 3 were also observed, and a small number of FJC(+)/ionized calcium-binding adaptor molecule 1 (Iba1)(+) microglia and FJC(+)/glial fibrillary acidic protein (GFAP)(+) astrocytes were observed in the normal brain. Next, we analyzed embryonic brains, in which the apoptosis of neural stem/precursor cells was induced by the administration of N-ethyl-N-nitrosourea (ENU) or ethanol at embryonic day 14 or 10, respectively. In those brains, FJC(+) neural stem/precursor cells and neuroepithelial cells expressing SRY-related HMG-box 2 (Sox2) were observed. Surprisingly degenerating mesenchymal cells were also FJC(+). The present study indicates that FJC is a reliable marker for degenerating neuronal cells during all differentiation stages. However, FJC could also label degenerating non-neuronal cells under some conditions.

Conserved and divergent expression dynamics during early patterning of the telencephalon in mouse and chick embryos.

The mammalian and the avian telencephalon are nearly indistinguishable at early embryonic vesicle stages but differ substantially in form and function at their adult stage. We sequenced and analyzed RNA populations present in mouse and chick during the early stages of embryonic telencephalon to understand conserved and lineage-specific developmental differences. We found approximately 3000 genes that orchestrate telencephalon development. Many chromatin-associated epigenetic and transcription regulators show high expression in both species and some show species-specific expression dynamics. Interestingly, previous studies associated them to autism, intellectual disabilities, and mental retardation supporting a causal link between their impaired functions during telencephalon development and brain dysfunction. Strikingly, the conserved up-regulated genes were differentially enriched in ontologies related to development or functions of the adult brain. Moreover, a differential enrichment of distinct repertoires of transcription factor binding motifs in their upstream promoter regions suggest a species-specific regulation of the various gene groups identified. Overall, our results reveal that the ontogenetic divergences between the mouse and chick telencephalon result from subtle differences in the regulation of common patterning signaling cascades and regulatory networks unique to each species at their very early stages of development.

Nonfluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

Analysis of Complete Neuroblast Cell Lineages in the Drosophila Embryonic Brain via DiI Labeling.

Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of an NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate individual cells within an NB clone, which reveals information about the temporal succession in which daughter cells are generated. Finally, we describe how to combine clonal DiI labeling with fluorescent antibody staining that permits relating protein expression to individual cells within a labeled NB lineage. These protocols make it feasible to uncover precise lineage relationships between a brain NB and its daughter cells, and to assign gene expression to individual clonal cells. Such lineage-based information is a critical key for understanding the cellular and molecular mechanisms that underlie specification of cell fates in spatial and temporal dimension in the embryonic brain.

Cellular stress mechanisms of prenatal maternal stress: Heat shock factors and oxidative stress.

Development of the brain prenatally is affected by maternal experience and exposure. Prenatal maternal psychological stress changes brain development and results in increased risk for neuropsychiatric disorders. In this review, multiple levels of prenatal stress mechanisms (offspring brain, placenta, and maternal physiology) are discussed and their intersection with cellular stress mechanisms explicated. Heat shock factors and oxidative stress are closely related to each other and converge with the inflammation, hormones, and cellular development that have been more deeply explored as the basis of prenatal stress risk. Increasing evidence implicates cellular stress mechanisms in neuropsychiatric disorders associated with prenatal stress including affective disorders, schizophrenia, and child-onset psychiatric disorders. Heat shock factors and oxidative stress also have links with the mechanisms involved in other kinds of prenatal stress including external exposures such as environmental toxicants and internal disruptions such as preeclampsia. Integrative understanding of developmental neurobiology with these cellular and physiological mechanisms is necessary to reduce risks and promote healthy brain development.

Exposure to Ionizing Radiation Triggers Prolonged Changes in Circular RNA Abundance in the Embryonic Mouse Brain and Primary Neurons.

The exposure of mouse embryos in utero and primary cortical neurons to ionizing radiation results in the P53-dependent activation of a subset of genes that is highly induced during brain development and neuronal maturation, a feature that these genes reportedly share with circular RNAs (circRNAs). Interestingly, some of these genes are predicted to express circular transcripts. In this study, we validated the abundance of the circular transcript variants of four P53 target genes (Pvt1, Ano3, Sec14l5, and Rnf169). These circular variants were overall more stable than their linear counterparts. They were furthermore highly enriched in the brain and their transcript levels continuously increase during subsequent developmental stages (from embryonic day 12 until adulthood), while no further increase could be observed for linear mRNAs beyond post-natal day 30. Finally, whereas radiation-induced expression of P53 target mRNAs peaks early after exposure, several of the circRNAs showed prolonged induction in irradiated embryonic mouse brain, primary mouse cortical neurons, and mouse blood. Together, our results indicate that the circRNAs from these P53 target genes are induced in response to radiation and they corroborate the findings that circRNAs may represent biomarkers of brain age. We also propose that they may be superior to mRNA as long-term biomarkers for radiation exposure.

The development of the serotonergic and dopaminergic systems during chicken mid-late embryogenesis.

Serotonin (5-HT) acts as a morphogen influencing embryonic brain development, and as a neurotransmitter regulating multiple biological functions with lifelong effects on animal physical, physiological and mental health, especially during the rapid growth phase prior to birth when embryos face many challenges to reach structural and functional completion. In this study, the development of the serotoninergic (5-HTergic) system and its modulatory effect on the dopaminergic (DAergic) system and related neural circuits were investigated during the mid-late embryogenesis, embryonic day (E)12-E20, in the chicken's brain. During 5-HTergic neuronal maturation, a growth-related anatomical and functional remodeling was highlighted: the 5-HT neurons continuously grew during E12-E20 except for a remarkable regression during E14-E16. Correspondingly, there was a time-dependent change in the 5-HT synthetic capacity. Specifically, 5-HT concentrations in the raphe nuclei increased from E12 to E14, reaching a first plateau during E14-E16, then continuously increased up to E19, and reaching a second plateau between E19-E20. The second plateau of the 5-HT concentration was in correspondence with the establishment of the 5-HTergic autoregulatory loop during E19-E20 and the development of the DAergic system. The DA concentrations remained unchanged from E12 to E16, then started to increase at E16, reaching a maximum at E19, and diminished before hatching. The unique developing time sequence between the 5-HTergic and DAergic systems suggests that the 5-HTergic system may play a critical role in forming the 5-HT - DA neural circuit during chicken embryogenesis. These results provide new insights for understanding the functional organization of the 5-HTergic system during embryonic development and raise the possibility that prenatally modulating the 5-HTergic system may lead to long-lasting brain structural and functional alterations.