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The vertebrate head and brain are characterized by highly complex morphological patterns. The forebrain, the most anterior division of the brain, is subdivided into the diencephalon, hypothalamus, and telencephalon from the neuromeric subdivision into prosomeres. Importantly, the telencephalon contains the cerebral cortex, which plays a key role in higher order cognitive functions in humans. To elucidate the evolution of the forebrain regionalization, comparative analyses of the brain development between extant jawed and jawless vertebrates are crucial. Cyclostomes – lampreys and hagfishes – are the only extant jawless vertebrates, and diverged from jawed vertebrates (gnathostomes) over 500 million years ago. Previous developmental studies on the cyclostome brain were conducted mainly in lampreys because hagfish embryos were rarely available. Although still scarce, the recent availability of hagfish embryos has propelled comparative studies of brain development and gene expression. By integrating findings with those of cyclostomes and fossil jawless vertebrates, we can depict the morphology, developmental mechanism, and even the evolutionary path of the brain of the last common ancestor of vertebrates. In this review, we summarize the development of the forebrain in cyclostomes and suggest what evolutionary changes each cyclostome lineage underwent during brain evolution. In addition, together with recent advances in the head morphology in fossil vertebrates revealed by CT scanning technology, we discuss how the evolution of craniofacial morphology and the changes of the developmental mechanism of the forebrain towards crown gnathostomes are causally related.

Prenatal exposure to environmental insults can increase the risk of developing neurodevelopmental disorders. Administration of the antiepileptic drug valproic acid (VPA) during pregnancy is tightly associated with a high risk of neurological disorders in offspring. However, the lack of an ideal human model hinders our comprehensive understanding of the impact of VPA exposure on fetal brain development, especially in early gestation. Herein, we present the first report indicating the effects of VPA on brain development at early stages using engineered cortical organoids from human induced pluripotent stem cells (hiPSCs). Cortical organoids were generated on micropillar arrays in a controlled manner, recapitulating the critical features of human brain development during early gestation. With VPA exposure, cortical organoids exhibited neurodevelopmental dysfunction characterized by increased neuron progenitors, inhibited neuronal differentiation and altered forebrain regionalization. Transcriptome analysis showed new markedly altered genes (e.g., KLHL1, LHX9, and MGARP) and a large number of differential expression genes (DEGs), some of which are related to autism. In particular, comparison of transcriptome data via GSEA and correlation analysis revealed the high similarity between VPA-exposed organoids with the postmortem ASD brain and autism patient-derived organoids, implying the high risk of autism with prenatal VPA exposure, even in early gestation. These new findings facilitate a better understanding of the cellular and molecular mechanisms underlying postnatal brain disorders (such as autism) with prenatal VPA exposure. This established cortical organoid-on-a-chip platform is valuable for probing neurodevelopmental disorders under environmental exposure and can be extended to applications in the study of diseases and drug testing. Miniaturized 3D models of the developing human brain provide a unique opportunity to assess the effects of a widely-used drug for neurological disorders on early-stage fetuses. Evidence suggests that valproic acid, a common treatment for epilepsy and bipolar disorder, may undermine healthy brain development in the first trimester of pregnancy. These effects have proven difficult to study, but researchers led by Jianhua Qin of the Dalian Institute of Chemical Physics have demonstrated that human cortical ‘organoids’ model offer valuable insights into this question. These stem cell-derived complex structures mirror key features of the fetal brain, by generating arrays of these organoids, the researchers could measure how valproic acid affects gene expression, cell growth, and maturation. They identified early signatures of abnormal development, supporting further use of this model to study the drug’s impact during pregnancy.

Background This study aimed to explore the molecular mechanism of cervical cancer (CC) by integrated bioinformatic analyses of gene expression and methylation profiles. Methods The gene expression and methylation microarrays in CC samples and normal controls were respectively downloaded from the GEO database. After screening the differentially expressed genes (DEGs) with Limma package and the CC-related methylation sites with CpGassoc package in R language, DEGs with CC-related methylation sites were identified from the intersection of the above two groups of results with 50 kb upstream and downstream of a gene as the gene region. Then GO enrichment was performed by GenCLIP2.0 software. Sequentially, analysis of metabolic sub-pathways with pathogenic risk was predicted by iSubpathwayMiner package in R language. Results A total of 1357 DEGs including 721 up-regulated and 636 down-regulated, as well as 666 CC-related methylation sites were screened out. After being analyzed, 26 DEGs with 35 CC-related methylation sites were identified. EDN3 and EDNRB were significantly involved in a function cluster in GO terms of vein smooth muscle contraction, vascular smooth muscle contraction and phasic smooth muscle contraction. LHX2 and PAX6 were significantly involved in a function cluster in GO terms of telencephalon regionalization and forebrain regionalization. ACOX3, CYP39A1 and DPYS were significantly enriched in 25 sub-pathways of 6 major pathways. Conclusions EDN3 and EDNRB might play important roles in the molecular mechanism of CC, and LHX2, ACOX3, CYP39A1 and DPYS might be susceptibility genes and potential risk markers in CC.

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay–Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup® Power 2.0 may be related to the improved efficacy in delivering glyphosate to cells, guaranteed by the specific surfactant formulation. In conclusion, the differences in GBH formulations should be carefully considered by the authorities, since sub-lethal and/or long-term effects (e.g. teratogenicity) can be significantly modulated by the active ingredient salt type and concentration of the adjuvants. Finally, the mechanistic toxicity of glyphosate and GBHs are worthy of further research.

International audience; In the current model, the most anterior part of the forebrain (secondary prosencephalon) is subdivided into the telencephalon dorsally and the hypothalamus ventrally. Our recent study identified a new morphogenetic unit named the optic recess region (ORR) between the telencephalon and the hypothalamus. This modification of the forebrain regionalization based on the ventricular organization resolved some previously unexplained inconsistency about regional identification in different vertebrate groups. The ventricular-based comparison also revealed a large diversity within the subregions (notably in the hypothalamus and telencephalon) among different vertebrate groups. In tetrapods there is only one hypothalamic recess, while in teleosts there are two recesses. Most notably, the mammalian and teleost hypothalami are two extreme cases: the former has lost the cerebrospinal fluid-contacting (CSF-c) neurons, while the latter has increased them. Thus, one to one homology of hypothalamic subregions in mammals and teleosts requires careful verification. In the telencephalon, different developmental processes between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish) have already been described: the evagination and the eversion. Although pallial homology has been long discussed based on the assumption that the medial-lateral organization of the pallium in Actinopterygii is inverted from that in Sarcopterygii, recent developmental data contradict this assumption. Current models of the brain organization are largely based on a mammalian-centric point of view, but our comparative analyses shed new light on the brain organization of Osteichthyes.

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)β signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFβ and SHH signaling. Stem Cells 2015;33:2496–2508

Understanding the factors that govern human forebrain regionalization along the dorsal-ventral and left-right (L-R) axes is likely to be relevant to a wide variety of neurodevelopmental and neuropsychiatric conditions. Recent work in lower vertebrates has identified several critical signaling molecules involved in embryonic patterning along these axes. Among these are the Wingless-Int (WNT) proteins, involved in the formation of dorsal central nervous system (CNS) structures, as well as in visceral L-R asymmetry. We examined the expression of WNT2b and WNT7b in the human brain, because these genes have highly distinctive expression patterns in the embryonic mouse forebrain. In the human fetal telencephalon, WNT2b expression appears to define the cortical hem, a dorsal signaling center previously characterized in mouse, which is also confirmed by BMP7 expression. In diencephalon, WNT2b expression is restricted to medial dorsal structures, including the developing pineal gland and habenular nucleus, both implicated in CNS L-R asymmetry in lower organisms. At 5 weeks gestation, WNT7b is expressed in cerebral cortical and diencephalic progenitor cells. As the cortical plate develops, WNT7b expression shifts, demarcating deep layer neurons of the neocortex and the hippocampal formation. Spatial and temporal expression patterns show startling similarity between human and mouse, suggesting that the developmental roles of these WNT genes may be highly conserved, despite the far greater size and complexity of the human forebrain.

The tissue, cellular and molecular mechanisms that regulate early regional specification of the vertebrate forebrain are largely unknown. We studied the expression patterns of Xbf-1, an anterior (and telencephalon) neural-specific winged helix transcription factor and Fgf-8, an early-secreted factor. This study looked at Xbf-1 and Fgf-8 expression in combination with embryonic grafting experiments and also used beads containing the recombinant Fgf-8 protein to determine these factors' effects upon forebrain patterning events. We provide evidence that additional Fgf-8 displaces Xbf-1 expression posteriorly, suggesting a concentration dependence of Fgf-8 for the early distinct regionalization of the telencephalic primordia. Also, additional stage 15 mid-anterior neural ridge (mANR) transplants inhibited telencephalon development, whereas lateral ANR transplants facilitated increased areas of telencephalon development. In both cases, these transplantations promoted ectopic expression of Xbf-1. These studies suggested that the distinct regionalization of the forebrain primordia involves the inhibitory actions of the mANR towards a telencephalon development and maintaining bilateral telencephali. These telencephalic primordia are initially localized by optimal Fgf-8 expression. The anterior mANR will eventually become the anterior and rostral diencephalic tissue. This in vivo study demonstrated Fgf-8 and the mANR are important in forebrain regionalization.

During the early steps of head development, ectodermal patterning leads to the emergence of distinct non-neural and neural progenitor cells. The induction of the preplacodal ectoderm and the neural crest depends onwell-studied signalling interactions between the non-neural ectoderm fated to become epidermis and the prospective neural plate. By contrast, the involvement of the non-neural ectoderm in the morphogenetic events leading to the development and patterning of the central nervous systemhas been studied less extensively. Here,we show that the removal of the rostral non-neural ectoderm abutting the prospective neural plate at late gastrulation stage leads, in mouse and chick embryos, to morphological defects in forebrain and craniofacial tissues. In particular, this ablation compromises the development of the telencephalon without affecting that of the diencephalon. Further investigations of ablated mouse embryos established that signalling centres crucial for forebrain regionalization, namely the axial mesendoderm and the anterior neural ridge, form normally. Moreover, changes in cell death or cell proliferation could not explain the specific loss of telencephalic tissue. Finally, we provide evidence that the removal of rostral tissues triggers misregulation of the BMP, WNT and FGF signalling pathways that may affect telencephalon development. This study opens new perspectives on the role of the neural/non-neural interface and reveals its functional relevance across higher vertebrates. ispartof: Development vol:141 issue:21 pages:4127-4138 ispartof: location:England status: published

Treatment of early gastrula- and neurula-staged Xenopus embryos with all- trans retinoic acid (RA) results in truncation of the anterior structures of the forebrain and head. The extent of truncation is dependent upon both the stage of immersion and the RA concentration used. As a method to investigate genes important during early forebrain regionalization, late gastrula and neurula embryos were immersed for 2 h within low (1 × 10 −9 M to 5 × 10 −8 M) concentrations of RA. Embryos were allowed to develop to tadpole stages and forebrain marker genes were assessed for any alteration in patterns of expression. Comparisons of controls to experimental groups indicated that the greatest sensitivity to low levels of RA occurred just before the initial expression of the forebrain-specific markers investigated. We concluded that forebrain regionalization and gene expression occurred in the following order: Xotx2 → Xsix3 → Xrx (& Xfez1 ) → Xbf1 → Xemx1 . Xsix3 seems to be very important for the initial parcellation of telencephalon, retinal and diencephalon areas.

The development of the central nervous system can be divided into a number of phases, each of which can be subject of genetic or epigenetic alterations that may originate particular developmental disorders. In recent years, much progress has been made in elucidating the molecular and cellular mechanisms by which the vertebrate forebrain develops. Therefore, our understanding of major developmental brain disorders such as cortical malformations and neuronal migration disorders has significantly increased. In this review, we will describe the major stages in forebrain morphogenesis and regionalization, with special emphasis on developmental molecular mechanisms derailing telencephalic development with subsequent damage to cortical function. Because animal models, mainly mouse, have been fundamental for this progress, we will also describe some characteristic mouse models that have been capital to explore these molecular mechanisms of malformative diseases of the human brain. Although most of the genes involved in the regulation of basic developmental processes are conserved among vertebrates, the extrapolation of mouse data to corresponding gene expression and function in humans needs careful individual analysis in each functional system., This work has been supported by the following grants: LSHG-CT-2004-512003, BFI2002-02979, BFU2005-09085, BFU2005-23722-E/BFI and IP from EU LSHG-CT-2004-512003. C. Vieira is a predoctoral fellow of the Gulbenkian PhD Program in Biomedicine supported by a grant from the Fundaçao para a Ciencia e a Tecnología do Ministerio para a Ciencia e o Encino Superior (FCT/MCES).

Pax6 is a highly conserved transcription factor essential for the development of the eyes in vertebrate and invertebrate species. It is also required for normal development of many regions of the central nervous system, including the mammalian forebrain, hindbrain and spinal cord. In the forebrain, it is expressed in a gradient in the dorsal telencephalon, where it is required for the expression of genes that confer dorsal characteristics and where it might play a role in regionalization of the cerebral cortex. It is expressed in the diencephalon, where it is essential for the specification of its derivatives. While the ancestral function of Pax6 may have been to specify a structure sensitive to light, it has been co-opted into the regulation of a broader range of processes in development of the vertebrate nervous system.

In situ hybridization was used to map spatiotemporal expression patterns of the four known intronless POU-III transcription factor genes Brn-1, Brn-2, Brn-4, and Tst-1 in the developing rat forebrain vesicle, beginning on embryonic day 10. The results indicate that the proliferation layers (ventricular and subventricular) and mantle layer of the forebrain neural tube each display a strikingly unique pattern of regionalized POU-III expression. Within a particular region, or layer within a region, none to all four of the mRNAs may be detected, and during development a particular mRNA in a particular region displays one of five expression patterns, or a combination of these patterns, which may be described as conserved, lost, transient, acquired, or redeployed expression. In the developing brain as a whole, Brn-1 and Brn-2 early on display somewhat different spatial expression patterns that converge to essential identity in the adult, whereas Brn-4 expression is initially broad and becomes much more restricted in the adult, and Tst-1 expression expands greatly through development. Usually, though not always, expression patterns tend to correlate with major histological features in the forebrain (often internal or external sulci associated with proliferation zones), and little evidence for waves of expression moving through the whole forebrain over time was obtained. Thus, clear differences in hybridization intensity often are observed between the cerebral cortex, basal telencephalic nuclei, hypothalamus, ventral thalamus, dorsal thalamus, and pretectal region. In contrast, transverse bands of hybridization extending from the roof to the floor of the forebrain, corresponding to proposed neuromeres, were not observed with these probes. The results suggest that POU-III transcription factors help define specific regions in the early neuroepithelium as well as different cellular phenotypes in the ventricular, subventricular, and mantle layers of specific regions later in development. Thus, the functions of these regulatory proteins may be different in proliferating neuroepithelial cells, young neurons, and mature neurons and appear to be region-specific.

Where do the regions of the forebrain come from? The paradigm that has come to light in the last ten years is that of a neural plate subdivided by soluble factors which (through the formation of gradients or of a molecular framework) transmits positional information to the neural stem cells. Some of those soluble factors are Fgf8, Sonic hedgehog and proteins of the BMP family. The neural stem cells interpret positional information in terms of the expression of combinations of certain transcription factors. Such combinations would finally be responsible for the formation of specific neural lineages (cortical, thalamic, etc.) from specific neuroepithelial regions. In addition, this primary positional information can make certain regions of the neural primordium (secondary organizers) secrete new soluble factors. These would in turn give rise to new positional information, spatially more restricted, so that the whole process would repeat itself to confer detail to a certain area. This wealth of new knowledge is already helping us to understand the cause of some brain malformations, and maybe we will soon be able to apply it to the early diagnosis and the prevention of such conditions.

Thereelingene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolutionin situhybridization analyses to determine the pattern ofreelinexpression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that expressreelintranscripts.reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelinwas detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages,reelinwas also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages,reelintranscripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelinexpression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.