Brain Patterning Research Articles

Downregulation of Pygopus 2 inhibits vascular mimicry in glioma U251 cells by suppressing the canonical Wnt signaling pathway.

Gliomas are the most common type of malignant primary brain tumor, and the Wnt signaling pathway is associated with glioma malignancy. Pygopus protein plays an important role in developmental brain patterning, and has been identified to be a component of the Wnt signaling pathway. In the present study, the Pygopus 2 (Pygo2) protein was examined in 80 glioma tissue samples. Short hairpin (sh)RNA-Pygo2 was transfected into glioma U251 cells, and the cell proliferation, colony formation and bromodeoxyuridine (BrdU) incorporation were analyzed. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect the expression of Pygo2. A vascular mimicry assay was performed to examine the vascular mimicry of U251 cells. A luciferase reporter assay was used to detect the β-catenin/Wnt system. The cyclin D1 protein was also detected using western blot analysis. The results demonstrated that inhibition of the expression of Pygo2 significantly triggered the decrease of cell proliferation, colony formation and BrdU incorporation compared with the cells treated with scramble control shRNA (shRNA-Scr). shRNA-Pygo2 transfection was found to inhibit vascular-mimicry and block the Wnt signaling pathway compared to the cells transfected with shRNA-Scr. The transfection of shRNA-Pygo2 also decreased the expression of the Wnt target gene cyclin D1. In conclusion, shRNA-Pygo2 suppressed glioma cell proliferation effectively and inhibited vascular mimicry by inhibiting the expression of cyclin D1 in the canonical Wnt/β-catenin pathway in brain glioma cells.

Distinct requirements for Wntless in habenular development

Secreted Wnt proteins play pivotal roles in development, including regulation of cell proliferation, differentiation, progenitor maintenance and tissue patterning. The transmembrane protein Wntless (Wls) is necessary for secretion of most Wnts and essential for effective Wnt signaling. During a mutagenesis screen to identify genes important for development of the habenular nuclei in the dorsal forebrain, we isolated a mutation in the sole wls gene of zebrafish and confirmed its identity with a second, independent allele. Early embryonic development appears normal in homozygous wls mutants, but they later lack the ventral habenular nuclei, form smaller dorsal habenulae and otic vesicles, have truncated jaw and fin cartilages and lack swim bladders. Activation of a reporter for β-catenin-dependent transcription is decreased in wls mutants, indicative of impaired signaling by the canonical Wnt pathway, and expression of Wnt-responsive genes is reduced in the dorsal diencephalon. Wnt signaling was previously implicated in patterning of the zebrafish brain and in the generation of left–right (L–R) differences between the bilaterally paired dorsal habenular nuclei. Outside of the epithalamic region, development of the brain is largely normal in wls mutants and, despite their reduced size, the dorsal habenulae retain L–R asymmetry. We find that homozygous wls mutants show a reduction in two cell populations that contribute to the presumptive dorsal habenulae. The results support distinct temporal requirements for Wls in habenular development and reveal a new role for Wnt signaling in the regulation of dorsal habenular progenitors.

Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

Dido mutations trigger perinatal death and generate brain abnormalities and behavioral alterations in surviving adult mice

Nearly all vertebrate cells have a single cilium protruding from their surface. This threadlike organelle, once considered vestigial, is now seen as a pivotal element for detection of extracellular signals that trigger crucial morphogenetic pathways. We recently proposed a role for Dido3, the main product of the death inducer-obliterator (dido) gene, in histone deacetylase 6 delivery to the primary cilium [Sánchez de Diego A, et al. (2014) Nat Commun 5:3500]. Here we used mice that express truncated forms of Dido proteins to determine the link with cilium-associated disorders. We describe dido mutant mice with high incidence of perinatal lethality and distinct neurodevelopmental, morphogenetic, and metabolic alterations. The anatomical abnormalities were related to brain and orofacial development, consistent with the known roles of primary cilia in brain patterning, hydrocephalus incidence, and cleft palate. Mutant mice that reached adulthood showed reduced life expectancy, brain malformations including hippocampus hypoplasia and agenesis of corpus callosum, as well as neuromuscular and behavioral alterations. These mice can be considered a model for the study of ciliopathies and provide information for assessing diagnosis and therapy of genetic disorders linked to the deregulation of primary cilia.

Endogenous gradients of resting potential instructively pattern embryonic neural tissue via Notch signaling and regulation of proliferation.

Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning in Xenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form of Notch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POU and HB4), directing undifferentiated cells toward neural fate in vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions between Vmem and key biochemical pathways (Notch and Ca(2+) signaling) as the molecular mechanism by which spatial differences of Vmem regulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects.

Modulating the expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics.

The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development.

Local and long-range endogenous resting potential gradients antagonistically regulate apoptosis and proliferation in the embryonic CNS.

Bioelectric signals, particularly transmembrane voltage potentials (Vmem), play an important role in large-scale patterning during embryonic development. Endogenous bioelectric gradients across tissues function as instructive factors during eye, brain, and other morphogenetic processes. An important and still poorly-understood aspect is the control of cell behaviors by the voltage states of distant cell groups. Here, experimental alteration of endogenous Vmem was induced in Xenopus laevis embryos by misexpression of well-characterized ion channel mRNAs, a strategy often used to identify functional roles of Vmem gradients during embryonic development and regeneration. Immunofluorescence analysis (for activated caspase 3 and phosphor-histone H3P) on embryonic sections was used to characterize apoptosis and proliferation. Disrupting local bioelectric signals (within the developing neural tube region) increased caspase 3 and decreased H3P in the brain, resulting in brain mispatterning. Disrupting remote (ventral, non-neural region) bioelectric signals decreased caspase 3 and highly increased H3P within the brain, with normal brain patterning. Disrupting both the local and distant bioelectric signals produced antagonistic effects on caspase 3 and H3P. Thus, two components of bioelectric signals regulate apoptosis-proliferation balance within the developing brain and spinal cord: local (developing neural tube region) and distant (ventral non-neural region). Together, the local and long-range bioelectric signals create a binary control system capable of fine-tuning apoptosis and proliferation with the brain and spinal cord to achieve correct pattern and size control. Our data suggest a roadmap for utilizing bioelectric state as a diagnostic modality and convenient intervention parameter for birth defects and degenerative disease states of the CNS.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration.

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Transient exposure to ethanol during zebrafish embryogenesis results in defects in neuronal differentiation: an alternative model system to study FASD.

BackgroundThe exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines.Methodology/Principal FindingsIn order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification.ConclusionOur investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.

Growth differentiation factor 11 is an encephalic regionalizing factor in neural differentiated mouse embryonic stem cells.

BackgroundThe central nervous system has a complex structural organization and consists of different subdomains along the antero-posterior axis. However, questions remain about the molecular mechanisms leading to the regionalization of this organ. We used a previously developed methodology to identify the novel patterning role of GDF11, a TGF-β signaling factor.FindingsUsing an assay based on neural differentiated mouse embryonic stem cells, GDF11 is shown to induce diencephalic (posterior forebrain), mesencephalic (midbrain) and metencephalic (anterior hindbrain) fates at the expense of telencephalic (anterior forebrain) specification. GDF11 has not previously been implicated in the early patterning of the nervous system. In addition, inhibition of the TGF-β type I receptors Alk4, Alk5 and Alk7 by the pharmacological inhibitor SB431542 caused a strong anteriorization of the cells.ConclusionsOur findings suggest that GDF11 is involved in the earliest steps of the brain patterning during neurogenesis in the vertebrate embryo and is shown to be a regionalizing factor of the regional fate in the developing brain. This regionalization is not a typical posteriorizing signal as seen with retinoic acid, FGF or BMP molecules. To our knowledge, this is the first time that GDF11 is implicated in the earliest steps of the patterning of the neural plate.

Conserved localization of Pax6 and Pax7 transcripts in the brain of representatives of sarcopterygian vertebrates during development supports homologous brain regionalization.

Many of the genes involved in brain patterning during development are highly conserved in vertebrates and similarities in their expression patterns help to recognize homologous cell types or brain regions. Among these genes, Pax6 and Pax7 are expressed in regionally restricted patterns in the brain and are essential for its development. In the present immunohistochemical study we analyzed the distribution of Pax6 and Pax7 cells in the brain of six representative species of tetrapods and lungfishes, the closest living relatives of tetrapods, at several developmental stages. The distribution patterns of these transcription factors were largely comparable across species. In all species only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum, and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, mainly in prosomeres 1 and 3. Pax7 specifically labeled cells in the optic tectum (superior colliculus) and Pax6, but not Pax7, cells were found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 labeling was detected in cells of the ventricular zone of the rostral alar plate and in migrated cells in the basal plate, including the griseum centrale and the interpeduncular nucleus. Caudally, Pax6 cells formed a column, whereas the ventricular zone of the alar plate expressed Pax7. Since the observed Pax6 and Pax7 expression patterns are largely conserved they can be used to identify subdivisions in the brain across vertebrates that are not clearly discernible with classical techniques.

Brain patterning perturbations following PTEN loss.

This review will consider the impact of compromised PTEN signaling in brain patterning. We approach understanding the contribution of PTEN to nervous system development by surveying the findings from the numerous genetic loss-of-function models that have been generated as well as other forms of PTEN inactivation. By exploring the developmental programs influenced by this central transduction molecule, we can begin to understand the molecular mechanisms that shape the developing brain. A wealth of data indicates that PTEN plays critical roles in a variety of stages during brain development. Many of them are considered here including: stem cell proliferation, fate determination, polarity, migration, process outgrowth, myelination and somatic hypertrophy. In many of these contexts, it is clear that PTEN phosphatase activity contributes to the observed effects of genetic deletion or depletion, however recent studies have also ascribed non-catalytic functions to PTEN in regulating cell function. We also explore the potential impact this alternative pool of PTEN may have on the developing brain. Together, these elements begin to form a clearer picture of how PTEN contributes to the emergence of brain structure and binds form and function in the nervous system.

Paraventricular Nucleus Sim1 Neuron Ablation Mediated Obesity Is Resistant to High Fat Diet

Single minded 1 (SIM1) is a transcription factor involved in brain patterning and control of energy balance. In humans, haploinsufficiency of SIM1 causes early-onset obesity. Mice deficient in the homologous gene, SIM1, also exhibit early onset obesity and increased sensitivity to a high fat diet. SIM1 is expressed in several areas of the brain implicated in control of energy balance including the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the medial amygdala and nucleus of the lateral olfactory tract. We have previously shown that mice with global Sim1 neuron ablation exhibit obesity with hyperphagia as the primary defect. The PVN has a critical role in feeding and in high-fat appetite, thus, we sought to determine the effect of Sim1 neuron ablation limited to the PVN. We achieved PVN-SIM1 limited ablation through stereotactic injection of diphtheria toxin into the PVN of Sim1Cre-iDTR mice. The specificity of this ablation was confirmed by immunohistochemistry and quantitative real time PCR of the PVN, supraoptic nucleus and the amygdala. Mice with PVN Sim1 neuron ablation, similar to mice with global Sim1 neuron ablation, exhibit early onset obesity with hyperphagia as the primary defect. However, PVN-Sim1 neuron ablated mice have a decreased response to fasting-induced hyperphagia. Consistent with this decrement, PVN-Sim1 neuron ablated mice have a decreased hyperphagic response to PVN injection of agouti-related peptide (AgRP). When PVN-Sim1 neuron ablated mice are placed on a high fat diet, surprisingly, their intake decreases and they actually lose weight. When allowed ad lib access to high fat diet and normal chow simultaneously, PVN-Sim1 neuron ablated mice exhibit overall decreased intake. That is, in PVN-Sim1 neuron ablated mice, access to fat suppresses overall appetite.

A chronological expression profile of gene activity during embryonic mouse brain development

The brain is a functionally complex organ, the patterning and development of which are key to adult health. To help elucidate the genetic networks underlying mammalian brain patterning, we conducted detailed transcriptional profiling during embryonic development of the mouse brain. A total of 2,400 genes were identified as showing differential expression between three developmental stages. Analysis of the data identified nine gene clusters to demonstrate analogous expression profiles. A significant group of novel genes of as yet undiscovered biological function were detected as being potentially relevant to brain development and function, in addition to genes that have previously identified roles in the brain. Furthermore, analysis for genes that display asymmetric expression between the left and right brain hemispheres during development revealed 35 genes as putatively asymmetric from a combined data set. Our data constitute a valuable new resource for neuroscience and neurodevelopment, exposing possible functional associations between genes, including novel loci, and encouraging their further investigation in human neurological and behavioural disorders.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-013-9486-7) contains supplementary material, which is available to authorized users.

Functional significance of cortical NMDA receptors in somatosensory information processing

N-methyl-d-aspartate receptor (NMDAR)-mediated activity is required for whisker-related neural patterning in the rodent brain. Deletion of the essential NMDAR subunit NR1 gene in excitatory cortical neurons prevents whisker-specific barrel formation and impairs thalamocortical afferent patterning. We used electrophysiological and voltage-sensitive dye imaging methods to assess synaptic and sensory evoked cortical activity and immunohistochemistry to examine immediate early gene expression following whisker stimulation in cortex-specific NR1 knockout (CxNR1KO) mice. In mutant mice, layer IV neurons lacked NMDAR-mediated excitatory postsynaptic currents, and temporal summation of excitatory postsynaptic potentials (EPSPs) was impaired. Barrel neurons showed both phasic and tonic responses to whisker deflection. The averaged tonic response in CxNR1KO mice was significantly less than that in control mice due to impaired EPSP temporal summation. Electrophysiological estimation of the number of thalamic neurons innervating single barrel neurons indicated a significant increase in CxNR1KO mice. Similarly, voltage-sensitive dye optical signals in response to whisker stimulation were widespread. Immediate early gene expression following whisker stimulation also showed a diffuse expression pattern in the CxNR1KO cortex compared with whisker-specific expression patterns in controls. Thus, when NMDAR function is impaired, spatial discrimination of whisker inputs is severely compromised, and sensory stimulation evokes diffuse, topographically misaligned activity in the barrel cortex.

High-Throughput, High-Frequency 3-D Ultrasound for in Utero Analysis of Embryonic Mouse Brain Development

With the emergence of the mouse as the predominant model system for studying mammalian brain development, in utero imaging methods are urgently required to analyze the dynamics of brain growth and patterning in mouse embryos. To address this need, we combined synthetic focusing with a high-frequency (38-MHz) annular-array ultrasound imaging system for extended depth-of-field, coded excitation for improved penetration and respiratory-gated transmit/receive. This combination allowed non-invasive in utero acquisition of motion-free 3-D data from individual embryos in approximately 2 min, and data from four or more embryos in a pregnant mouse in less than 30 min. Data were acquired from 148 embryos spanning 5 d of early to mid-gestational stages of brain development. The results indicated that brain anatomy and cerebral vasculature can be imaged with this system and that quantitative analyses of segmented cerebral ventricles can be used to characterize volumetric changes associated with mouse brain development.

Dvr1 transfers left–right asymmetric signals from Kupffer's vesicle to lateral plate mesoderm in zebrafish

An early step in establishing left–right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the left lateral plate mesoderm, activating a transcriptional response of Nodal expression in the left lateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the left LPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFβ family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left–right signal from KV to the LPM.

Perturbations in cortical development and neuronal network excitability arising from prenatal exposure to benzodiazepines in mice

During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.

Neural progenitors, patterning and ecology in neocortical origins

The anatomical organization of the mammalian neocortex stands out among vertebrates for its laminar and columnar arrangement, featuring vertically oriented, excitatory pyramidal neurons. The evolutionary origin of this structure is discussed here in relation to the brain organization of other amniotes, i.e., the sauropsids (reptiles and birds). Specifically, we address the developmental modifications that had to take place to generate the neocortex, and to what extent these modifications were shared by other amniote lineages or can be considered unique to mammals. In this article, we propose a hypothesis that combines the control of proliferation in neural progenitor pools with the specification of regional morphogenetic gradients, yielding different anatomical results by virtue of the differential modulation of these processes in each lineage. Thus, there is a highly conserved genetic and developmental battery that becomes modulated in different directions according to specific selective pressures. In the case of early mammals, ecological conditions like nocturnal habits and reproductive strategies are considered to have played a key role in the selection of the particular brain patterning mechanisms that led to the origin of the neocortex.

Xnr3 affects brain patterning via cell migration in the neural-epidermal tissue boundary during early Xenopus embryogenesis

Neural induction and anteroposterior neural patterning occur simultaneously during Xenopus gastrulation by the inhibition of BMP and Wnt signaling, respectively. However, other processes might be necessary for determining the neural-epidermal boundary. Xenopus nodal-related-3 (Xnr3) is expressed in dorsal blastula and plays a role in neural formation. In this study, we analyzed how Xnr3 affects neural patterning to identify novel mechanisms of neural-epidermal-boundary determination. In situ hybridization revealed that ventro-animal injection with Xnr3 shifted the lateral krox20 expression domain anteriorly and reduced Otx2 expression. The mature region of Xnr3 is necessary for these effects to occur, and the pro-region accelerated them. Phalloidin labeling revealed that cells around the neural-epidermal boundary lost their slender shape following Xnr3 injection. Moreover, we analyzed the cell migration of ectodermal cells and found specific Xnr3-induced effects at the neural-epidermal boundary. These findings together suggested that Xnr3 affects anterior ectoderm migration around the neural-epidermal boundary to induce a specific neural pattern abnormality. Change of the shape of surrounding ectodermal cells and the specific migratory pattern might therefore reflect the novel mechanism of neural-epidermal boundary.